Table of Contents - Scripps Research Institutepva.scripps.edu/files/pdf/PVA2013.pdf · Table of Contents Table of Contents ... Architecture of a viral capsid in complex with the maturation

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Table of Contents

Table of Contents ................................................................................... 1

XXIII Biennial Conference on Phage/Virus Assembly ............................ 2

Conference at a Glance .......................................................................... 3

Meeting Schedule ................................................................................... 4

Poster Presentations ............................................................................ 12

Speaker Abstracts ................................................................................. 15

Poster Abstracts ................................................................................... 95

VIPERdb .............................................................................................. 132

Previous Meetings .............................................................................. 142

Attendee Listing ................................................................................. 143

Acknowledgements ............................................................................ 149

Sponsors ............................................................................................. 150

Map ........................................................................................ back cover

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XXIII Biennial Conference on Phage/Virus Assembly

UCLA Lake Arrowhead Conference Center & Mountain Resort Lake Arrowhead, CA 8-13 September 2013

We are delighted that you have joined us for the 23rd biennial Phage Virus Assembly Meeting. We are pleased that we can convene at the site of the first meeting in 1968 and that the organizer of the first meeting joins us to give a historical perspective of this venerable gathering. Our meeting this year is broadly represented by a strong mix of senior scientists who have been regularly participating in these meetings and of junior researchers for whom this is their first PVA. This spectrum involves a nice age distribution ranging from graduate students, post docs, and freshly minted assistant professors to those of us that are not so young. We are also happy to have such a strong European contingent and that the next PVA will be held in Switzerland. For many of us this meeting represents the best of science gatherings – open exchange of scientific wisdom, data and reagents, as well as the making and renewing of long lasting, warm, friendships. We are clearly standing on the shoulders of those who had the early foresight to see the common themes in virology regardless of virus and host (e.g. Jon King, Roger Burnett and Peter Prevelige) and who made the effort to expand this gathering from phage virology in its early years to include the full range of viruses. These initiatives also lead to the FASEB Virus Assembly Meetings that alternate with PVA and provide a perspective of virology beyond the particle. In spite of its breadth, we feel that PVA is still a highly focused meeting that provides an exceptional opportunity for those of us interested in the morphogenesis of virus particles and their biological behaviors to share our results. When Max Perutz was asked why there were so many Nobel Prizes awarded to researchers at the LMB in Cambridge, he thought for a moment and said, “I think it is the canteen”. We hope that this meeting can be the “canteen” for some great new virology.

Organizing Committee: Bill Gelbart, Jack Johnson

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XXIII Biennial Conference on Phage/Virus Assembly – Conference at a Glance

Sunday 8 September

2:00 PM Registration Begins 6:00 PM Dinner 8:00 PM Opening Session Session 1

Monday 9 September

9:00 AM Entry/Exit Session 2 10:45 AM Assembly Session 3 3:00 PM Poster Session A 8:00 PM Structure I Session 4

Tuesday 10 September

9:00 AM Terminase I Session 5 10:45 AM Genome Replication Session 6 8:00 PM Structure and Assembly I Session 7

Wednesday 11 September

9:00 AM Terminase II Session 8 10:30 AM Structure and Assembly II Session 9 3:00 PM Poster Session B 8:00 PM Structure and Assembly III Session 10

Thursday 12 September

9:00 AM Structure II Session 11 10:45 AM Packaging I Session 12 2:00 PM Structure III Session 13 3:45 PM Packaging II Session 14 6:30 PM Special Dinner

Friday 13 September 9:00 AM Departure

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September 8-13, 2013 XXIII Biennial Conference on Phage/Virus Assembly

Sunday 8 September

2:00 PM Registration begins at UCLA Conference Center 6:30 – 8:00 PM Dinner 8:00 PM OPENING SESSION Chairs: Bill Gelbart and

Jack Johnson Fred Eiserling UCLA Reflections on the first PVA meetings Jonathan King MIT From Arrowsmith to self-assembly, through gene splicing and protein folding to climate change

Monday 9 September

8:00 – 9:00 AM Breakfast 9:00 – 10:30 AM ENTRY/EXIT Chair: Michael Rossmann Brian Bothner Montana State Characterization of lipase activation during adeno-associated virus entry Nicola Gerardo Abrescia CIC bioGUNE, Spain Electron tomography study of PRD1 during DNA translocation Nichole Cumby University of Toronto More than a simple ruler: Defined regions of the bacteriophage tape measure dictate host entry requirements Senjuti Saha University of Toronto Tails that kill: Characterization of the bactericidal activity of P. aeruginosa F-type pyocins Petr Leiman EPFL Contractile ejection systems and their central spike proteins Ian Molineux University of Texas at Austin Cryo-electron tomography of phage infection

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10:30 – 10:45 AM Coffee Break 10:45 AM – 12:00 NOON ASSEMBLY Chair: James Conway David Veesler TSRI Architecture of a viral capsid in complex with the maturation protease Terje Dokland University of Alabama at Birmingham Molecular piracy and the mobilization of Staphylococcus aureus pathogenicity islands Kamel El Omari University of Oxford Plate tectonics of virus shell assembly and reorganization in phage Φ8 Nicole Steinmetz Case Western Reserve University Structure-function studies of plant virus-based cargo-delivery systems Rees Garmann UCLA Reconstituted plant viral capsids can release genes to mammalian cells 12:00 – 1:00 PM Lunch 3:00 PM POSTER SESSION A 6:30 – 8:00 PM Dinner 8:00 PM STRUCTURE I Chair: Carolyn Teschke Vijay S Reddy TSRI Structure and organization of cement proteins in Human adenovirus James F. Conway University of Pittsburgh Herpesvirus capsids visualized at near atomic resolution Susan Schroeder University of Oklahoma Advancing viral RNA structure prediction Alexis Huet University of Pittsburgh Structural basis of T5 capsid expansion and stabilization Chi-yu Fu TSRI Atomic structure of the 75MDa extremophile Sulfolobus turreted icosahedral virus determined by cryoEM and X-ray crystallography

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William Klug UCLA What drives conformational strain in viruses? Preeti Gipson Baylor College of Medicine Capsid proteins of an ancient marine virus violates all local symmetries in an icosahedral particle

Tuesday 10 September

8:00 – 9:00 AM Breakfast 9:00 AM – 10:30 AM TERMINASE I Chair: Paulo Tavares Zachary Berndsen UCSD Non-equilibrium dynamics and heterogeneity in phi29 DNA packaging Zhengyi Zhao University of Kentucky Discovery of a new motion mechanism of biomotors analogous to the earth revolving around the sun without rotation Douglas Smith UCSD Structural basis for force generation by the T4 viral DNA packaging motor Shelley Grimes University of Minnesota The role of pRNA in the DNA packaging motor of bacteriophage phi29 Lindsay W. Black University of Maryland Bacteriophage T4 terminase translocates on a “crunched” A form-like DNA Song Gao The Catholic University of America Mechanism of DNA packaging initiation by the small and large terminase proteins of bacteriophage T4 10:30 – 10:45 AM Coffee Break 10:45 AM – 12:00 NOON GENOME REPLICATION Chair: Peter Stockley Andrew Routh TSRI Deep sequencing the RNA encapsidated by viruses: mapping of RNA recombination; discovery of functional motifs; and packaging of host RNAs including retrotransposons Odisse Azizgolshani UCLA Inhibition of primary translation of the viral genome is the dominant mechanism of super-infection exclusion in Sindbis virus

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Pooja Saxena John Innes Centre Unraveling the tangled processes of RNA replication and encapsidation in Cowpea mosaic virus Bentley Fane University of Arizona Regulating the switch between (ds) replicative form and (ss) genomic DNA biosynthesis: old adversaries and new genes Sarah Doore University of Arizona Particle morphogenesis: the major barrier to horizontal gene exchange between microviruses 12:00 – 1:00 PM Lunch 1:00 – 6:30 PM Free Time 6:30 – 8:00 PM Dinner 8:00 PM STRUCTURE AND ASSEMBLY I Chair: Ian Molineux James A. Geraets University of York Using a Hamiltonian paths approach to analyze the RNA organization observed in cryo-electron tomograms of pilus-bound MS2 Aaron Roznowski University of Arizona Structure-function analysis of the phiX174 DNA pilot protein, prefabricated on-site tail assembly Cameron Haase-Pettingell MIT Horned cyanophage Syn5 assembly proceeds through a scaffolding and portal containing procapsid Alasdair Steven NIAMS-NIH Visualizing the internal structure of bacteriophage T7 by bubblegram imaging Francoise Livolant CNRS Why do pauses happen during bacteriophage DNA ejection? Lei Sun University of Purdue Conserved infection mechanism of bacterial viruses: ΦX174 forms a tail for DNA transport Gino Cingolani Thomas Jefferson University A pH-dependent conformational switch in the tail needle gp26 triggers genome ejection

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Wednesday 11 September

8:00 – 9:00 AM Breakfast 9:00 – 10:15 AM TERMINASE II Chair: Doug Smith Marc Morais University of Texas Medical Branch, Galveston Structural insights into the bacteriophage Φ29 dsDNA packaging motor Juan Luis Loredo-Varela University of York X-ray structure of the small terminase protein from the thermophilic bacteriophage G20C Philomena Ostapchuk Stony Brook University Role of the adenovirus core protein VII in virus assembly, genome packaging and gene expression Paul Jardine University of Minnesota Coordination and communication in a complex biological nanomotor Karen L. Maxwell University of Toronto A phage-encoded HNH protein is required for HK97 terminase function 10:15 – 10:30 AM Coffee Break 10:30 – 12:00 NOON STRUCTURE AND ASSEMBLY II Chair: Peter Prevelige Jason Perlmutter Brandeis University Viral genome structures are optimal for capsid assembly Aida Llauro University of Madrid Mechanical stability and reversible fracture of vault particles Sergey Venev University of Massachusetts Medical School Segment self-repulsion is the major driving force of influenza genome packaging Guido Polles SISSA Predicting functional units in viral capsids from quasi-rigid capsid subdivisions Mark Young Montana State University STIV ATPase and genome structure Elizabeth Pierson Indiana University Late Intermediates in the Assembly of Virus Capsids

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12:00 – 1:00 PM Lunch 3:00 PM POSTER SESSION B 6:30 – 8:00 PM Dinner 8:00 PM STRUCTURE AND ASSEMBLY III Chair: Jon King Andreas Kuhn University of Hohenheim Assembly of single coat proteins of filamentous phage in the membrane of Escherichia coli Tina Motwani University of Connecticut Getting the kinks out: Unravelling the role of bacteriophage P22’s spine helix during capsid maturation Michael Hagan Brandeis University Membrane assisted virus assembly Bonnie Oh University of Pittsburgh The delta domain of bacteriophage HK97 is required for capsid assembly and portal incorporation Guillaume Tresset CNRS Norovirus capsid proteins self-assemble through biphasic kinetics via long-lived stave-like intermediates Tatiana Domitrovic TSRI Capsid maturation of a non-enveloped animal virus enables subunit-specific membrane lytic activity and generates hysteresis in structural transitions controlled by pH Chuan Hong Baylor College of Medicine CryoEM of portal machinery of bacteriophage PRD1

Thursday 12 September

8:00 – 9:00 AM Breakfast 9:00 – 10:30 AM STRUCTURE II Chair: Alasdair Steven Kristin Parent Michigan State University Shigella outer membrane proteins A & C mediate Sf6 genome ejection Peng Ge UCLA Atomic model of the pyocin reveals how energy is stored to “kill”

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Sherwood R Casjens University of Utah The phage P22 tail needle affects DNA delivery Al Katz City College of New York

Cryo-EM Single Particle Reconstruction of P2 and P7 Proteins of Cystovirus φ6 Mohamed Zairi CNRS Gp12: a viral collagen-like protein that binds to the bacteriophage SPP1 capsid Steve Harvey Georgia Tech An Atomistic Model for Bacteriophage MS2 and Implications for Viral Assembly 10:30 – 10:45 AM Coffee Break 10:45 – 12 NOON PACKAGING I Chair: Chuck Knobler Dave Bauer Carnegie Mellon University Herpesvirus genomes, the pressure is on Alex Evilevitch Carnegie Mellon University Solid to fluid DNA phase-transition inside the virus. Genome metastability Eric Dykeman University of York More than passive passengers: The roles of RNA packaging signals in virus assembly and evolution Peter G. Stockley University of Leeds Packaging signals in ssRNA viruses from bacteria to plants to humans Li Dai The Catholic University of America Real-time single molecule fluorescence analysis shows that one inactive mutant subunit in a pentameric bacteriophage T4 DNA packaging motor can be tolerated 12:00 – 1:00 PM Lunch

2:00 – 3:30 PM STRUCTURE III Chair: Karen Maxwell Michael Rossmann Purdue University Structure of a dengue virus temperature-induced fusogenic intermediate Amelie Leforestier CNRS DNA confined inside the bacteriophage capsid. What can we learn from cryo-EM? Abhay Kotecha University of Oxford Structure and dynamics of picornavirus to inform vaccine design

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Shuji Kanamaru Tokyo Institute of Technology The crystal structure and the stability of C-terminal fragment of gp34, proximal half of long tail fiber of bacteriophage T4 Thomas Smith Danforth Plant Science Center Atomic structure of cucumber necrosis virus and the role of the capsid in vector transmission Robert L. Duda University of Pittsburgh An atomic model of the T=9 procapsid of phage D3 3:30 – 3:45 PM Coffee Break 3:45 – 5:15 PM PACKAGING II Chair: Bob Duda Venigalla B. Rao The Catholic University of America Delivery of vaccine genes and proteins into mammalian cells using the bacteriophage T4 DNA packaging machine Wen Jiang Purdue University Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7 Philip Serwer University of Texas Health Science Center at San Antonio DNA length quantization in phage T3/T7 DNA packaging and expulsion Paulo Tavares CNRS A mechanistic view of bacteriophage headful packaging Michael Feiss University of Iowa DNA packaging by phage N15 Roman Tuma, University of Leeds Shedding light on the early stages of RNA packaging by single molecule fluorescence

6:30 – 8:00 PM Dinner

Friday 13 September

8:00 – 9:00 AM Breakfast Departure

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Poster Sessions

Session A - Monday 9 September 3:00 PM

A.1 Nina Atanasova University of Helsinki Characterization of archaeal spindle-shaped virus His1 and its comparison to other short-tailed spindle-shaped viruses A.2 Dennis Bamford University of Helsinki Cryo-EM of Portal Machinery of Bacteriophage PRD1 A.3 Nina Broeker University of Potsdam The interaction of phage HK620 with its LPS receptor on host cell surface A.4 Pascale Boulanger CNRS Maturation of T5 capsid: a view throughout in vitro expansion and decoration A.5 Patricia Campbell University of Pittsburgh Comparing the common structural subunits of phage phi1026b and HK97 by high resolution cryo-electron microscopy A.6 Wei Dai Baylor College of Medicine Visualizing virus assembly intermediates inside marine cyanobacteria A.7 Damian delToro UCSD Mechanisms of Termination of Bacteriophage DNA Packaging Studied with a Single-molecule Optical Tweezers Measurement A.8 Andrei Fokine Purdue University The molecular architecture of the bacteriophage T4 neck A.9 Ramesh Goel University of Utah Bacteriophages in engineered bioreactors and natural systems-phage bacteria interactions A.10 Boon Chong Goh University of Illinois at Urbana-Champaign Capsid-locking mechanism in the maturation of a T=4 virus A.11 Paul Gottlieb City College of New York Virus-like particles assembled for use as respiratory virus vaccines A.12 Sandra Greive University of York Investigating the DNA binding activity of the DBD of the small terminase protein (GP1) from SPP1.

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A.13 Roger Hendrix University of Pittsburgh On the mechanism of phage HK97 capsid protein crosslinking and a novel bacteriophage A.14 Christian Beren UCLA Determining the end-to-end distance of long RNA molecules A.15 Surendra W. Singaram UCLA Simple Statistical Mechanical Theory for the Yield of RNA Packaging by CCMV Capsid Protein A.16 Devin Brandt UCLA Development of an agrobacterial system for growing CCMV virus in cowpea plants, for studying RNA packaging in planta, and for mutating capsid protein for functionalization of CCMV virus-like particles A.17 Radhika Gopal TSRI

Analysis of nodaviral genome packaging during mixed infection of BHK cells A.18 Michael DiMattia NIH Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein

Session B - Wednesday 11 September 3:00 PM

B.1 Rosie Hill University of Alabama at Birmingham Derepression and encapsidation of Staphylococcus aureus pathogenicity islands by helper phages B.2 Nhung Huynh TSRI Perspectives on the Crystal Structure of Human Adenovirus B.3 Cathy Yan Jin UCLA

Pressure-dependent genome ejections of double-stranded DNA viruses B.4 Bradley Kearney TSRI

CryoEM-based geometric and dynamic analysis of Nudaurelia capensis ω virus maturation reveals the energy landscape of particle transitions B.5 Nicholas Keller UCSD Effect of Spermidine on Bacteriophage phi29 DNA Packaging

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B.6 Thomas Klose Purdue University The structure of R135, a fiber associated enzyme of Mimivirus B.7 Outi Leena Lyytinen University of Helsinki Insights into the Pseudomonas phage phi6 envelope assembly through expression of the component proteins in Escherichia coli B.8 Reginald McNulty TSRI Elucidating the Structure and Mechanism of Bacteriphage P22 Genome Packaging B.9 ChoonSeok Oh University of Iowa Mutations of putative Walker A and B motifs and Loop-Helix-Loop motif in large subunit of terminase affect Phage Lambda DNA packaging B.10 James Short TSRI The cellular distribution of (+) RNA during Flock House virus infection: Implications for the selection of genomic RNA during virus assembly B.11 Jean Sippy University of Iowa Genetic Characterization of the Proposed Q and Coupling Motifs in the Packaging ATPase of Bacteriophage Lambda B.12 Carolyn Teschke University of Connecticut Incorporation of portal protein in in vitro assembled P22 procapsids B.13 Mariska van Rosmalen Vrije University Testing the strength of adenovirus by single particle AFM nanoindentation B.14 Sergey Venev University of Massachusetts Medical School Virion packaging and release constrain intrahost sequence diversity of influenza A virus B.15 Moh Lan Yap Purdue University Structural Studies of Bacteriophage T4 Baseplate by X-ray Crystallography B.16 Eric May University of Connecticut Molecular Simulation Study of the Gating Mechanism of the Lassa Virus Nucleoprotein

B.17 Sari Mäntynen University of Jyväskylä Characterization of ΦN - A fresh water bacteriophage resembling cystoviruses B.18 Lu Xie Carnegie Mellon University Fitting simulation models to viral capsid assembly systems

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Speaker Abstracts

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Reflections on the first PVA meetings

Fred Eiserling

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From Arrowsmith to Self-Assembly, through Gene Splicing

and Protein Folding to Climatic Change.

Jonathan A. King1

1 Dept of Biology, MIT, Cambridge, MA 02139

The early interest in bacteriophages reflected excitement over a potential new form of anti-bacterial therapy. However when the development of the electron microscope reveled the common icosahedral lattices among the capsids of animal, plant and bacterial viruses, phage emerged as a model for understanding virus structure and infection. The development of temperature sensitive (ts) and amber conditional lethal mutations by Robert S. Edgar and Richard H Epstein opened up the direct study of the intracellular assembly of bacteriophage. Laemmli’s high resolution SDS gel system led to a leap forward in the molecular biochemistry of these and many other processes. The counter-intuitive discovery that capsid assembly proceeded through a preformed shell, which was subsequently filled with DNA, led to the first lambda vectors for introducing foreign genes into bacteria and gene splicing technology. Of course the earlier characterization of restriction enzymes provided the enzymological basis for genetic engineering. With the discovery of GroE a new dimension of protein folding and assembly was revealed. Further studies of temperature sensitive assembly mutants revealed that many were defective in protein folding, opening up analysis of the intracellular folding of complex proteins, and the failure of such processes leading to inclusion bodies. Continuing investigations of phage structure provided a key platform for the advancement of cryo Electron Microscopy technologies. The relative recent recognition of the importance of marine photosynthetic cyanobacteria in the global carbon cycle, and the recognition of the world wide oceanic distribution of marine phages, suggests a new dimension, through understanding and manipulation of cyanobacterial photosynthetic processes.

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Characterization of Lipase Activation During

Adeno-Associated Virus Entry

Vamseedhar Rayaprolu1, Navid Movahed1, Dewey Brooke1, Ravi Kant1, Balasubramanian Venkatakrishnan2, Bridget Lins2, Antonette Bennett2, Robert McKenna2, Mavis Agbandje-

McKenna2, and Brian Bothner1

1 Department of Chemistry and Biochemistry, Montana State University, Bozeman MT, USA. 2 Department of Biochemistry and Molecular Biology and Center for Structural Biology, The McKnight Brain Institute, University of Florida Gainesville, FL, USA.

Adeno-associated virus (AAV) is a small-icosahedral virus that contains a phospholipase domain within the capsid protein. The phospholipase A2 (PLA2) domain is believed to be packaged inside the capsid during assembly, becoming externalized during endocytosis. Currently, the trigger(s) for activation and functional aspects of the PLA2 domain during endocytosis are unclear. We are using a number of biochemical and biophysical approaches to study AAV capsid protein stability, dynamics, and receptor binding with respect to PLA2 externalization and endcytosis. To this end we have developed a sensitive lipase assay for tracking activity. EM analysis shows that lipase activity leads to tubule formation on liposomes, suggesting a role in serotype specific entry pathways.

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Electron tomography study of PRD1 during DNA translocation

Peralta B.1, Gil-Carton D.1, Castaño-Díez D.2, Bertin A.3, Boulogne C.3, Oksanen HM.4, Bamford

DH4, Abrescia NG1,5

1 Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio, Spain. 2 Center for Cellular Imaging and Nano-Analitics (C-CINA) Biozentrum, University of Basel, Mattenstrasse 26 CH-4058 Basel. 3 Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université de Paris-Sud, 91 405 ORSAY Cedex. 4 Institute of Biotechnology and Department of Biosciences, Viikki Biocenter, University of Helsinki, P.O. Box 56, Viikinkaari 5, 00014 University of Helsinki, Finland 5 IKERBASQUE, Basque Foundation for Science, 48011 Bilbao, Spain

PRD1 is an internal membrane-containing bacteriophage that infects Gram-negative cells. A wealth of biochemical and structural information has been accumulated on PRD1 during the past 20 years. So far it remains the only phage with a membrane whose icosahedral structure has have been visualized at 4.2Å by X-ray crystallography elucidating fundamental aspects of viral evolution. During infection to deliver its genome across the cellular membrane, the PRD1 membrane transforms to a tubular structure protruding from one of the twelve vertices. Using a combination of electron microscopy techniques we show that this nanotube exits from the same unique vertex used for DNA packaging, that it is structured and that its nucleation is triggered by changes in osmolarity. We also snapshot this proteo-lipidic tube in action, tunneling through the gram-negative bacterial cell envelope of infected Salmonella enterica and Escherichia coli. Our findings unveil the mechanism behind the genome translocation across the cell envelope.

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More than a simple ruler: Defined regions of the bacteriophage tape measure dictate host entry requirements

N. Cumby1, A. M. Edwards1, A. R. Davidson1,2, and K. L. Maxwell1.

1 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 2 Department of Biochemistry, University of Toronto, Toronto, ON, Canada

Bacteriophages are obligate parasites that propagate within their bacterial hosts. In order to replicate, the phage must first inject its DNA into the host bacterium. Phage-host interactions begin with the phage tail fiber binding to the bacterial outer membrane receptor. A conformational change triggers DNA ejection from the head, and it traverses through the phage tail, passing through the bacterial periplasm and inner membrane to its final destination in the cytosol. While this process is very poorly understood, the phage tape measure protein (TMP) is known to play an important role. The TMP is required for assembly of siphophage tails, with the tail tube protein assembling around the length of the TMP. It is also required for cell entry, and is expelled from the phage tail upon infection and believed to associate with the membrane[1]. Previous studies revealed that internal deletions of the TMP result in phages with shorter tails [2,3]. Most of these deletions were competent for tail assembly, but defective for DNA injection [2]. To gain insight into the requirement of specific TMP protein sequences for DNA injection, we are using the E. coli siphophage HK97 to examine host/phage interactions. We discovered that HK97 requires two host proteins, the periplasmic chaperone, FkpA and the inner membrane protein PtsG for DNA entry. By engineering phages with hybrid TMPs, we have been able to show that the requirements for these proteins can be bypassed by inserting TMP sequences from related phages that are not dependent on them. In addition, we are able to engineer dependence on these factors by inserting regions of the HK97 TMP into other related phages that normally do not require FkpA or PtsG. These results illustrate that TMP sequence specifies periplasmic and inner membrane host protein requirements.

[1] Roessner, C. A., Ihler, G. M. J. Bact. 157, 165 (1984) [2] Katsura, I. Nature. 327, 73 (1987) [3] Katsura, I. Adv. Biophy. 26, 1 (1990)

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Tails that Kill: Characterization of the Bactericidal Activity of P. aeruginosa F-type Pyocins

Senjuti Saha1, Alan Davidson1,2, and Karen Maxwell1.

1 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada 2 Department of Biochemistry, University of Toronto, Toronto, ON, Canada

Approximately 95% of phages have a tail to mediate host cell interaction and serve as the conduit for nucleic acid injection during infection. Interestingly, Pseudomonas aeruginosa strains naturally produce specialized phage tail-like molecules, called R- and F-pyocins, that are lethal to other P. aeruginosa strains and related species. Previous bioinformatic studies revealed conservation of gene order and sequence homology of the R- and F-type pyocins to tail proteins from Myoviridae and Siphoviridae phages respectively. The similarities between the naturally occurring pyocins and phage tails suggested that any isolated phage tail might possess bactericidal activity. To test this hypothesis, we isolated tails from P. aeruginosa siphophage DMS3, E. coli siphophage λ, and E. coli myophages Mu and P2. These tails all lacked bactericidal activity, implying that tail-like pyocins possess properties unique from phage tails that allow them to kill bacteria. To gain a greater understanding of the differences between F-pyocins and siphophage tails, we used iterative PSI-BLAST and HHPred searches to characterize each open reading frame in the F-pyocin operon. While there is significant sequence similarity between most of the tail structural proteins, including the tail tube, tape-measure, and tail tip proteins, we identified three open reading frames at the 3’ end of the F-pyocin operon that lack homology to any annotated phage protein. We have determined that these genes are essential for the assembly of F-type pyocins. In addition, we have shown that specificities of F-type pyocins may be redirected by genetically switching their side tail fiber proteins and we are investigating the receptors that these pyocins bind to on their target cells. We have been able to show that LPS on the cell surface may act as a shield or a receptor for F-type pyocins, like in the case of R-type pyocins.

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Contractile ejection systems and their central spike proteins

Petr Leiman1

1 École Polytechnique Fédérale de Lausanne (EPFL), Switzerland Contractile tails of bacteriophages and related systems - R-type pyocins, the Serratia entomophila antifeeding prophage, the Photorhabdus Virulence Cassette, and the Type VI Secretion System (T6SS) - contain a special spike-shaped protein complex, which is involved in breaching the target cell envelope during infection. We have identified the genes and determined crystal structures for several spike proteins from phages, pyocins, and T6SS, and established a paradigm for their organization and function. The architecture of spike proteins is remarkably well conserved at the level of tertiary structure, but the corresponding genes and amino acid sequences have undergone huge rearrangements with domains becoming separate genes that are very far away from each other in the genome. Large bacteriophages and T6SS have the most complex spikes, in which the tip is a small protein that forms a very sharp conical extension on the spike. This membrane-attacking tip is stabilized by a buried Fe ion in all phage and pyocin spikes, and with a Zn ion in T6SS spikes at an equivalent position. Remarkably enough, the Fe and Zn binding sites are formed by the same residues in the same configuration, and the reason for the selection of one metal ion in favor of the other is unclear.

The spike tip proteins belong to the PAAR (Proline-Alanine-Alanine-aRginine) repeat domain superfamily, which currently includes several thousand members in the GenBank. PAAR repeat proteins from T6SS are often extended by a domain with a putative effector function (nuclease, DNases, peptidases, etc.) or by a transthyretin domain. PAAR knockout mutants of Vibrio cholerae and Acinetobacter baylyi have either reduced or completely abolished T6SS activity, showing that PAAR proteins are essential for T6SS function and can play an important role in building of the T6SS machine or target cell membrane piercing or both. A unique HMM profile of PAAR repeat proteins makes it possible to identify the spike tip proteins in many phages including T4, phiKZ, P1, etc. Complete structures (including the tip protein) of phage T4 central spike and T6SS spike of Vibrio cholerae will be presented and discussed.

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Cryo-electron tomography of phage infection

Jiagang Tu1, Bo Hu1, Xiaowei Zhao1, Jun Liu1, Ian J. Molineux2.

1 Dept. Pathology, University of Texas Medical School, Houston, Texas, USA 2 Molecular Genetics and Microbiology, University of Texas, Austin, Texas

Phages are intrinsically stable organisms, and as such must be triggered to initiate infection. After adsorption a number of sequential events are associated with triggering; the phage must penetrate the outer membrane of a Gram-negative cell, traverse the periplasm, passing through the peptidoglycan cell wall, and then breach the cytoplasmic membrane, forming a channel for DNA ejection into the cytoplasm. For myophages and podophages, triggering is associated with extensive structural remodelling of the infecting particle. Using high-throughput cryo-electron tomography, we have captured structural intermediates at unprecedented resolution in the infection process [1]. We can now begin to understand the different strategies used by tailed phages in penetrating the Gram-negative cell envelope.

[1] B. Hu, W. Margolin, I. J. Molineux, and J. Liu. Science. 339:576-579 (2013)

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Architecture of a dsDNA viral capsid in complex with its maturation protease

D. Veesler1, R. Khayat1, S. Krishnamurthy2, J. Snijder3,4, R.K. Huang1, A.J.R. Heck3,4, G.S.

Anand2 and J.E. Johnson1

1 The Scripps Research Institute, 10550 N. Torrey Pines Road MB-31. La Jolla, CA 92037. USA 2Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore. 3Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences Utrecht University, Padualaan 8, 3584CH, Utrecht, The Netherlands. 4Netherlands Proteomics Centre Padualaan 8, 3584CH, Utrecht, The Netherlands.

Most dsDNA viruses, including bacteriophages and herpesviruses, rely on a staged assembly process of capsid formation [1]. A viral protease is required for many of them to disconnect scaffolding domains/proteins from the capsid shell, therefore priming the maturation process [2]. We used the bacteriophage HK97 as a model system to decipher the molecular mechanisms underlying the recruitment of the maturation protease by the assembling procapsid and the influence exerted onto the latter. Comparisons of the procapsid with and without protease using single-particle electron cryomicroscopy reconstructions, hydrogen/deuterium exchange coupled to mass spectrometry and native mass spectrometry demonstrated that the protease interacts with the scaffolding domains within the procapsid interior and stabilizes them as well as the whole particle. The results suggest that the thermodynamic consequences of protease packaging are to shift the equilibrium between isolated coat subunit capsomers and procapsid in favor of the latter by stabilizing the assembled particle before making the process irreversible through proteolysis of the scaffolding domains. [1] D. Veesler, J.E. Johnson. Annu Rev Biophys. 41:473-96 (2012). [2] R.K. Huang, R. Khayat, K.K. Lee, I. Gertsman, R.L. Duda, R.W. Hendrix, J.E. Johnson. 408:541-554 (2011)

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Molecular piracy and the mobilization of Staphylococcus aureus pathogenicity islands

Terje Dokland1, Rosie Hill1, Keith Manning1, Cynthia M. Rodenburg1,

Jamil S. Saad1, Erin A. Wall2, Gail E. Christie2

1Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 2Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA

Molecular piracy is a process in which one replicon (the “pirate”) usurps the replication and assembly process of an unrelated viral replicon for its own propagation [1]. Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements that encode virulence factors such as superantigen toxins. Normally repressed and stably integrated into the host genome, SaPIs become derepressed and mobilized by specific “helper” phages, resulting in packaging of the SaPI genome into transducing particles made of structural proteins encoded by the helper. In many cases, the capsids formed in the presence of the SaPIs are smaller (T=4) than those produced by the phage alone (T=7) [2, 3]. This capsid size redirection is dependent on two SaPI-encoded proteins, CpmA and CpmB [4]. CpmB is a dimer that acts as an internal scaffolding protein [3]. SaPIs also encode their own small terminase subunit, which causes specific packaging of SaPI DNA into the capsids. This process of piracy is reminiscent of that found in the bacteriophage P2/P4 system, where the plasmid replicon P4 packages its own genome into small capsids formed by proteins supplied by a P2 helper. In the P2/P4 system, however, the size redirection is dependent on a single P4-encoded protein, Sid, which forms an external scaffold on the P4 procapsids. Thus, the pirate replicons in each case have evolved distinct mechanisms for changing the size of their respective helpers and interfering with phage multiplication [1]. [1] Christie, G.E. and Dokland. T. (2012). Pirates of the Caudovirales. Virology 434, 210-221. [2] Spilman, M.S., Dearborn, A.D., Chang, J.R., Damle, P.K., Christie, G.E., Dokland, T. (2011) A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids. J. Mol. Biol. 405, 863-876. [3] Dearborn, A.D., Spilman, M.S., Damle, P.K., Chang, J.R., Monroe, E.B., Saad, J.S., Christie, G.E., Dokland, T. (2011) The Staphylococcus aureus pathogenicity island 1 protein gp6 functions as an internal scaffold during capsid size determination. J. Mol. Biol. 412, 710-722 [4] Damle, P.K., Wall, E.A., Spilman, M.S., Dearborn, A.D., Ram, G., Novick, R.P., Dokland, T., Christie, G.E. (2012). The roles of SaPI1 proteins gp7 (CpmA) and gp6 (CpmB) in capsid size determination and helper phage interference. Virology 432, 277-282.

26

Plate tectonics of virus shell assembly and reorganization in

phage Φ8

Kamel El Omari1

1 University of Oxford

The hallmark of a virus is its capsid, which harbours the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a unique arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity. Finally the oligomeric organization of the subunits in the crystal and solution suggest an unexpected assembly pathway which proceeds via a pentameric intermediate.

27

Structure-function studies of plant virus-based cargo-

delivery systems

Sourabh Shukla1, Amy M. Wen1, Karin L. Lee1, Michael A. Bruckman1, Nicole F. Steinmetz1,2,3,4

1 Department of Biomedical Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA. 2 Department of Radiology, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA. 3 Department of Materials Science and Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA. 4 Department of Macromolecular Science and Engineering, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106, USA.

The in vivo fate, i.e. biodistribution and clearance, of nanomaterials is dependent on their physiochemical properties such as shape, size, and surface chemistry. For nanomedical applications, precise formulation of homogeneous and monodispersed materials in thus an important goal. Nature has already perfected the self-assembly of various nanostructured molecules and materials; we have turned toward the structures of plant viruses (termed viral nanoparticles, VNPs). These highly symmetrical nanomaterials come in various shapes and sizes, but each species is highly monodisperse. Viruses naturally evolved to develop cargos to specific cells and tissues. These features render VNPs attractive cargo-delivery systems for medical applications. In this presentation, we will highlight structure-function based studies specifically characterizing tissue-specificity of VNPs of varying geometries, i.e. size, flexibility, aspect ratio. We will discuss self-assembly protocols that allow shape switching of VNP rods to spheres, facilitate the synthesis of VNP rods of varying but defined aspect ratios, and self-assembly of co-operative VNP networks and chains.

28

Reconstituted plant viral capsids can release genes to

mammalian cells

Rees F. Garmann1, Odisse Azizgolshani1, Ruben Cadena-Nava2, Charles M. Knobler1, and

William M. Gelbart1 1 Department of Chemistry and Biochemistry, University of California, Los Angeles CA 90095 1569 USA 2 Universidad Nacional Autónoma de México, Centro de Nanociencias y Nanotecnología, Ensenada B.C. 22860, Mexico. The nucleocapsids of many plant viruses are significantly more robust and protective of their RNA contents than those of enveloped animal viruses, making them an attractive option for virus-based gene delivery research. In particular, the capsid protein (CP) of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is of special interest because it has been shown to spontaneously package, with high efficiency, a large range of lengths and sequences of single-stranded RNA molecules. We demonstrate that hybrid virus-like particles, assembled in vitro from purified CCMV CP and RNA replicons containing a heterologous gene of interest, are capable of disassembly when delivered to the cytoplasm of mammalian cells. Following this disassembly step and release of the RNA cargo, the RNA undergoes replication and translation resulting in high levels of expression of the gene of interest. These results establish the first steps in the use of plant viral capsids as vectors for gene delivery and in situ protein expression in mammalian cells. Furthermore, the CCMV capsid protects the packaged RNA against nuclease degradation and serves as a robust external scaffold with many possibilities for further functionalization and cell targeting.

29

Structure And Organization Of Cement Proteins In Human Adenovirus

Vijay S. Reddy1 and Glen R. Nemerow2

1 Department of Integrative Structural and Computational Biology, 2 Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

Replication defective and conditionally replicating adenoviruses (AdV) vectors are currently being utilized in ~25% of human gene transfer clinical trials. Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. In particular, there exists a significant ambiguity with regards to the identity and location of the four cement proteins (IIIa, VI, VIII and IX) that stabilize the contiguous capsid shell and their role in mediating the interactions between the major capsid proteins. The recently determined X-ray structure of a human adenovirus (HAdV) vector at near atomic resolution represents a milestone as the largest biomolecular structure yet determined using X-ray diffraction methods[1]. The crystal structure revealed detailed interactions between the major capsid proteins and characteristic structural features of several accessory molecules that stabilize the AdV capsids. Recently, the refined crystal structure of HAdV has provided greater structural insights, which facilitated the revision of the previous assignments of several cement proteins derived from the cryoEM studies. The details of these new findings will be discussed. These new details unveiled by the refined crystal structure of HAdV represents a significant step forward in understanding the structural underpinnings of AdV assembly and cell entry mechanisms of a large dsDNA virus and provides new opportunities for improving adenovirus mediated gene transfer.

[1] Reddy VS, Natchiar SK, Stewart PL, Nemerow GR. Crystal structure of human adenovirus at 3.5 A resolution. Science. 2010;329(5995):1071-5.

30

Herpesvirus capsids visualized at near atomic resolution

J.F. Conway,1 J.D. Yoder,1 A.M. Makhov,1 H.R. Lopez,1 J.B. Huffman,2

F.L. Homa2 & M. Vos3

1 Dept. of Structural Biology, University of Pittsburgh School of Medicine, USA. 2 Dept. of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, USA. 3 Apps Lab, FEI Europe B.V., Eindhoven, The Netherlands.

The canonical capsid protein fold of the bacteriophage HK97 appears likely to be common to all dsDNA tailed bacteriophages. Consequently, similarities abound in capsid morphologies as well as in the processes of assembly, DNA packaging and maturation. We recently had the opportunity to use a new-generation FEI Falcon II direct-electron detection camera to collect high-resolution cryo-electron microscope images of bacteriophages D3, phi1026b and T5 (see Poster by Duda et al., Campbell et al. and Huet et al., respectively) from which we calculated structures that provide fascinating insights into the flexibility of the HK97 fold. In addition, also acquired copious cryoEM images of herpesvirus capsids, including capsids in virions and B-capsids from the cell nucleus. Our goal is to understand common features of capsid architecture and assembly as well as the structural factors governing capsid size and organization. The complexity of the herpesvirus capsid compared to those of bacteriophages presents considerable challenges in structural analysis. Four major capsid proteins and two essential minor proteins are arrayed on the icosahedral herpesvirus capsid in copy numbers from 60 to 960 and with masses from 12 to 150 kDa, whereas the HK97, D3, phi1026b and T5 capsids comprise a single major capsid protein of ~45 kDa in the procapsid and ~32 kDa after maturation. The herpesvirus capsid mass is >200 MDa, but atomic models are available for only 2 fragments of the capsid structural proteins – the upper domain of the 150 kDa VP5 major capsid protein, and the C-terminal three-quarters of the 63 kDa UL25 protein that forms a heterodimer with pUL17 that is called the capsid vertex specific component (CVSC). Nonetheless, we are able to successfully fit the VP5 upper domain model into our ~6 Å resolution cryoEM map as a solid body and reproduce many of the features of the crystal structure. We can also fit the main elements of the HK97 capsid protein fold into the lower domain of the herpesvirus capsid, as has been done previously. We are, however, unable to accommodate the pUL25 model which appears to be more compact that the cryoEM density indicates. One striking feature of the virion capsid that has not been previously observed is the presence of tubes of density arrayed as five pairs on the inner surface of the capsid and radiating out from the pentamers. We see no such density under the hexamers, but infer that it is most likely a portion of the N-terminal domain of the VP5 major capsid protein and may serve to stabilize the pentamers against the internal pressure of the packaged genome. We continue to analyze the structural data as we attempt to further refine the density, and expect to be able to compare the virion capsid structure with the B-capsid structure at the same resolution.

31

Advancing Viral RNA Structure Prediction

Susan J. Schroeder1, Jonathan W. Stone1, Samuel Bleckley1, Jui-Wen Lui1

1 University of Oklahoma, 101 Stephenson Boulevard, Norman, OK 73019 USA

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and assembled into viral particles. Thus, viral RNA poses many interesting RNA folding challenges. Multiple solutions to the RNA genome folding problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold. Recent improvements in computational methods are presented to explore the possible secondary structures for encapsidated Satellite Tobacco Mosaic Virus RNA [1], MS2 bacteriophage RNA [2], and Alfalfa Mosaic Virus RNA [3]. The diverse landscape of RNA conformational space includes many canyons and crevices distant from the lowest minimum free energy valley that remain unexplored by traditional RNA structure prediction methods. The Crumple algorithm provides a method to efficiently compute all possible secondary structures for a given RNA sequence without consideration of thermodynamics. Traditional free energy minimization methods do not consider stabilizing RNA tertiary interactions, RNA-protein interactions, or the possibility that kinetics rather than thermodynamics determines the functional structures. Crumpling an RNA sequence, like crumpling a piece of paper, is a fast and indiscriminate way of folding. Experimental filters make the complete enumeration of all possible structures for an RNA sequence a reasonable approach. Experimental filters from chemical or enzymatic probing, phylogenetic covariation, SELEX, crystallography, or cryoelectron microscopy can reduce conformational space without overlooking structures that may be stabilized by tertiary and quaternary interactions. Chemical probing does not necessarily define a single structure. The minimum number and length of helices has a significant effect on reducing conformational space and has inspired the next version of Crumple. For example, the combined effect of all filters reduces the possible number of structures for an Alfalfa Mosaic Virus protein binding sequence from over 50 million structures to a set of 91 structures. The predictions from this new approach can test hypotheses and models of viral assembly and guide construction of complete three-dimensional models of virus particles. Recent alternative models of in vitro and encapsidated STMV RNA use free energy minimization [4, 5]. The similarities and differences between these models and the ensemble model will be discussed. The new models highlight the fundamental question of whether kinetics or thermodynamics drives virus assembly.

[1]. S.J. Schroeder, J.W. Stone, S. Bleckley, T. Gibbons, D.M. Mathews, Biophys. J., 101. 167-175 (2011).

[2]. S. Bleckley, S.J Schroeder. RNA 18. 1309-1318 (2012). [3]. S. Bleckley, J.W. Stone, S.J. Schroeder, S.J. PLoS ONE, 7. e52414 (2012). [4]. E.J.S. Archer, N.J. Watts, R. O'Kane, B. Wang, D.A. Erie, A. McPherson, K.M. Weeks, K. M. 52.

3182-90 (2013). [5]. S.S.G. Athavale, J. C. Bowman, N.V. Hud, L.D. Williams, S.C. Harvey, S. C.PLoS ONE 8. e54384 (2013).

32

Structural basis of bacteriophage T5 capsid expansion and

stabilization

Alexis Huet1,2, Pascale Boulanger3, Robert Duda2, Roger Hendrix2, James F. Conway1 1 University of Pittsburgh School of Medicine, Biomedical Science Tower 3, Pittsburgh, PA 15260 U.S.A. 2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. 3 IBBMC- CNRS UMR8619, Bât 430 - Université Paris-Sud, F-91405 Orsay, France.

The capsid assembly of tailed dsDNA bacteriophages is a regulated process that involves several steps. The phage DNA is pumped into a preformed compact procapsid which, in turn, is subject to a major reshaping that yields a thinner and expanded shell. This capsid expansion is a conserved feature that leads to a bigger (twofold increases of its internal volume) and more stable capsid. In this work, we focus on the capsid of the bacteriophage T5, a model of a large icosahedral (T=13) capsid. 775 subunits of the major head protein (pb8) and 12 subunits of the portal protein (pb7) are required to make up the final structure. Neither chemical crosslinking nor decoration proteins are required for final capsid stability, as shown recently [1]. In this work, we obtained structures of purified prohead II and empty expanded capsid at subnanometer resolution by cryo-EM and image reconstruction. The reshaping of the structural subunit during expansion is observed at a molecular level, allowing us to characterize the interactions and conformational changes that occur during capsid maturation.

[1] Preux O, Durand D, Huet A, Conway JF, Bertin A, Boulogne C, Drouin-Wahbi J, Trévarin D, Pérez J, Vachette P, Boulanger P. J Mol Biol. 425, 1999 (2013)

33

Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray

crystallography

David Veesler1, Thiam-Seng Ng2,3, Anoop K. Sendamarai4,5, Brian J. Eilers4,5, C. Martin Lawrence4,5, Shee-Mei Lok2,3 Mark J. Young5,6, John E. Johnson1, and Chi-yu Fu1

1 Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 2 Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, Singapore 169857 3 Center for Bioimaging Sciences, National University of Singapore, Singapore 169857 4 Department of Chemistry and Biochemistry, 5 Department of Plant Sciences and Plant Pathology, 6 Department of Thermal Biology Institute, Montana State University, Bozeman, MT 59717

Sulfolobus Turreted Icosahedral Virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of trans-membrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses suggesting that they derive from a common ancestor predating the divergence of the three Kingdoms of life. One architectural motif (β-jelly roll fold) forms virtually the entire capsid (distributed in 3 different gene products) indicating that a single ancestral protein module may have been at the origin of its evolution [1].

[1] Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5504-9.

34

What Drives Conformational Strain in Viruses?

L.E. Perotti1, W.S. Klug1, J. Rudnick2 and R.F. Bruinsma2

1 Mechanical and Aerospace Engineering Department, UCLA, Los Angeles, CA, USA 2 Physics and Astronomy Department, UCLA, Los Angeles, CA, USA

The notion of quasi-equivalence introduced by Caspar and Klug has provided an explanation of how multiple copies of the same protein can occupy positions of different symmetry environments on an icosahedral viral shell by adopting local conformational strains, The implicit assumption of quasi-equivalence is that such strains are small, and are driven by quaternary structure. However, an increasing number of viruses are being discovered to undergro structural transitions characterized by large conformational strains, Moreover, in some cases, notably the well-studied bacteriophage HK97, these conformational strains are driven by enthalpic interactions that act on capsomers even in solution, independent of assembled-shell quaternary structure. We develop a theory of conformational strain in icosahedral capsids founded upon an extended version of continuum elasticity that accounts for the discreteness of capsomer boundaries and use our model to evaluate the effects of icosahedral quaternary structure and internal enthalpic forces on the emergent patterns of conformational strain and global capsid morphology. We find that both mechanisms can drive global conformational strain in general, but that the nature and pattern of the strains depends crucially on icosahedral T-number. In particular, we predict that for a certain class of T-numbers enthalpically-driven conformational strain would necessarily break icosahedral symmetry.

35

Capsid Proteins of an Ancient Marine Virus Violate All Local Symmetries in an Icosahedral Particle

Preeti Gipson1, Matthew L. Baker1, Xiangan Liu1, Desislava Raytcheva2, Cammie Haase3,

Jacqueline Piret2, Jonathan A. King3 and Wah Chiu1 1 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030 USA; 2 Department of Microbiology, Northeastern University, Boston, MA 02115 USA 3 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA

Despite being the most abundant life forms in the oceans, marine viruses and their impact on our global ecosystems are not fully understood. Cyanophages, which infect cyanobacteria such as Synechococcus and Prochlorococcus, are critical for host genetic diversity and microbial community variability. Here, we report the capsid structure of a dsDNA marine virus, Syn5, from electron cryo-microscopy and modeling. Its capsid structure deviates from the quasi-equivalent icosahedral packing, revealing a unique arrangement of novel outer capsid proteins, present along a specific diagonal of hexameric capsomeres protruding from the capsid surface. This positioning of outer capsid proteins breaks all local two/three-fold symmetry interfaces within and across the capsomeres of the icosahedral shell. Such an arrangement has not been observed in other known phage/virus structures. Furthermore, our analysis of Syn5 procapsids suggests that these protruding densities incorporate during virus maturation, enhancing capsid stability and efficiency. Here, the observation of an icosahedral capsid packing, at the energetic cost of local symmetry breaks in evolutionary ancient marine viruses (~2.8 billion years), highlights the significance of virus capsid evolution paving the way for highly efficient pathogens.

36

Nonequilibrium Dynamics and Ultra-slow Relaxation of Confined DNA During Viral Genome Packaging

Zachary T. Berndsen1, Nicholas Keller1, Paul J. Jardine 2, Shelly Grimes2, and Douglas Smith E.

Smith1

1 University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, United States 2 University of Minnesota, 18-246 Moos Tower, 515 Delaware Street SE, Minneapolis, MN 55455, United States

Many viruses utilize molecular motors that generate large forces to package DNA to high densities. The nature of the DNA dynamics and forces resisting confinement is a subject of wide debate and is of interest as a model for understanding the physics of confined polymers. Here we show that DNA in bacteriophage phi29 undergoes nonequilibrium conformational dynamics during packaging with a relaxation time >60,000× longer than for free DNA and >3000× longer than reported for DNA confined in nanochannels. Nonequilibrium dynamics significantly increases the load on the motor, causes heterogeneity in the packaging rates of individual viruses, and explains the frequent pausing observed in motor translocation. Similar effects may play a role in many other biological processes involving confined biomolecules and active transport by motors.

37

Discovery of a New Motion Mechanism of Biomotors Analogous to the Earth Revolving around the Sun without

Rotation

P. Guo1, C. Schwartz, J. Haak1, and Z. Zhao1

1 Nanobiotechnology Center, and Markey Cancer Center, Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA

Biomotors have been classified into linear and rotational motors. It has been popularly believed that viral dsDNA-packaging apparatuses are pentameric rotation motors. Recently, a third class of hexameric motor [1-3] has been described in bacteriophage phi29 that utilizes a mechanism of revolution without rotation, friction, coiling, or torque [4]. It addresses here that how the packaging motor controls dsDNA one-way traffic [5]; how four electropositive layers in the channel interact with the electronegative phosphate backbone to generate four steps in translocating one dsDNA helix; how the motor resolves the mismatch between 10.5 bases and 12 connector subunits per cycle of revolution; and how ATP regulates the sequential action of motor ATPase [6]. This revolution mechanism helps resolve puzzles and debates concerning the oligomeric nature of the packaging motor in many phage systems since motors with all numbers of subunits can utilize the revolution mechanism that solves the undesirable dsDNA supercoiling issue during translocation.

[1] C. Schwartz, G. M. De Donatis, H. Fang, P. Guo, Virology (2013). [2] Y. Shu, F. Haque, D. Shu, W. Li, Z. Zhu, M. Kotb, Y. Lyubchenko, P. Guo, RNA 19, 766 (2013). [3] H. Zhang, J. Endrizzi, Y. Shu, F. Haque, C. Sauter, L. Shlyakhtenko, Y. Lyubchenko, P. Guo, Y.

Chi, RNA In press (2013). [4] C. Schwartz, G. M. De Donatis, H. Zhang, H. Fang, P. Guo, Virology (2013). [5] Z. Zhao, E. Khisamutdinov, C. Schwartz, P. Guo, ACS Nano (2013). [6] C. Schwartz, H. Fang, L. Huang, P. Guo, Nucleic Acids Res. 40, 2577 (2012).

38

Structural basis for force generation by the T4 viral DNA packaging motor

Amy Davenport Migliori1, Nicholas A. Keller1, Tanfis I. Alam2, Marthandan Mahalingam2,

Venigalla B. Rao2, Douglas E. Smith1

1 Dept. of Physics, University of California, San Diego, Mail Code 0379, 9500 Gilman Drive, La Jolla, CA, 92093 USA 2 Dept. of Biology, The Catholic University of America, 620 Michigan Ave. NE, Washington, DC, 20064 USA

Viral DNA packaging motors generate enormous forces to package DNA into capsids at high densities [1-4]. Recent crystallographic and cryo-EM structures of the phage T4 gp17 DNA packaging motor protein suggest that DNA translocation may be driven by gp17 ratcheting between extended and compact conformations, in which the transition is orchestrated by electrostatic interactions between complimentarily charged residues across the interface between N- and C-terminal domains [5]. We tested this model by a combination of molecular dynamics simulations, single-molecule optical tweezers measurements, and site-directed mutagenesis. Our calculations show that the free energy difference between the extended and compact states is indeed sufficient to generate the large measured forces. In addition, we find that electrostatic interactions contribute significantly to this free energy difference, although hydrophobic interactions are equally important. To further test the model, we study the effect of mutations altering charged residues predicted to destabilize the compact state. We find strong correlation between calculated changes in free energy and measured impairments of motor function. Together, our findings support the proposed gp17 ratchet model and yield insights into the mechanism of force generation by the T4 motor. [1] Smith DE, et al. Nature 413(6857): 748-752 (2001) [2] Fuller DN, Raymer DM, Kottadiel VI, Rao VB & Smith DE. Proc Natl Acad Sci U S A 104(43): 16868-

16873 (2007) [3] Fuller DN, et al. J Mol Biol 373(5): 1113-1122 (2007) [4] Smith DE. Current Opinion in Virology 1(2): 134-141 (2011) [5] Sun S, et al. Cell 135(7): 1251-1262 (2008)

39

The Role of pRNA in the DNA Packaging Motor of Bacteriophage ø29

Shelley Grimes1, Wei Zhao1, Sheng Cao1, Wei Zhang1, Marc Morais2,

and Paul Jardine1 1 Diagnostic and Biological Sciences, University of Minnesota, Minneapolis, MN 55455 USA 2 Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 USA During assembly, double-stranded DNA bacteriophages package their genomes into the viral head, compacting the DNA to near-crystalline density. This process is driven by powerful ATP-dependent molecular motors that assemble on the portal vertex. In ø29, the motor is comprised of three ring-shaped complexes: the dodecameric head-tail connector gp10, the pentameric ring of prohead RNA (pRNA), and the ring ATPase gp16. While the pRNA component is unique to ø29 and its relatives, the challenges encountered in DNA packaging are similar amongst the dsDNA phages, suggesting that the essential functions provided by pRNA are likely encoded in subdomains of the packaging proteins of other phages. Here we report recent insights into motor assembly derived from an integrated approach to probe the structure/function role of pRNA in the motor: 1) For other dsDNA phages, the large terminase subunit (packaging ATPase) assembles directly to the head, while in ø29 pRNA is the motor component that interacts and binds to the head. The pRNA makes contacts with both the connector and the head shell, inducing a conformational change in the shell subunits surrounding the portal vertex. The ATPase then assembles upon this RNA scaffold; 2) For other dsDNA phages, the small terminase subunit mediates genome recognition. While ø29 has no exact equivalent of the small terminase, we have identified a new functional domain in pRNA that mediates orientation of DNA packaging and genome recognition; and 3) The ø29 motor has been shown to be highly coordinated, implying a high degree of communication within the motor. The centrally located position of pRNA in the motor suggests a potential role as mediator of communication. Here the intermolecular interaction between pRNAs that serves to “lock” the pRNA ring on the head during assembly may also play a role in communication between motor subunits and between the connector and ATPase during packaging.

40

Bacteriophage T4 terminase translocates on a “crunched” A form-like DNA

L.W. Black1, A.B. Dixit1, J.A. Thomas1, and K. Ray1

1 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore MD 21201 USA Viral genome packaging into capsids is powered by ATP driven high-force-generating motor proteins. The phage T4 terminase large subunit gp17 packages linear (60bps-170kbps) DNAs of any sequence efficiently in vitro without the small terminase subunit gp16. Bulky fluorescent dyes attached to the DNA ends or bases are packaged efficiently with the DNAs into portal containing proheads. However DNA base intercalating dyes (ID) are highly inhibitory to packaging. Rare terminase mutations (only three such sites) confer resistance to IDs. These mutations probably involve amino acids at the interface between the motor and the DNA at the N-terminal ATPase domain. ATP-powered translocation in vitro expels all detectable tightly bound YOYO-1 ID dye from packaged short dsDNA substrates [1]. FRET distance measurements strongly suggest that the C-terminal nuclease domain of the T4 terminase docks to the prohead portal clip region [2], a docking orientation that is not consistent with a packaging model in which the N-terminal ATPase domain of the T4 terminase binds to the prohead portal clip region [3]. In stalled Y-DNAs, FRET has shown a decrease in distance from the terminase C-terminus to the portal in the stalled Y-DNA conformation. DNA B compression to near A form (33% distance decrease B to A expected) is supported in motor stalled Y-stem DNAs by FRET increase between close dye pairs. In addition there is complete removal of YOYO-1 (which NMR shows not to bind to A form) from DNA during translocation. These studies support a proposed "DNA crunching" or compression motor mechanism [4] involving a transient structural change in B form DNA to A form-like during translocation. Transient compression of B DNA to A form during packaging and movement of terminase subunits toward the portal shows striking similarities to a mechanism and structure proposed recently for the DnaB hexameric helicase ATP powered dsDNA translocation on A form DNA [5]. We previously proposed in a “synapsis model” that the terminase gp16 small subunit binds together two pac site containing DNA sequences in order to gauge concatemer maturation and regulate initiation of packaging [6]. The model is supported by genetics and purified protein structural work showing gp16 single and double side by side protein-only rings (proposed to be non-planar lock washer-like structures). Surprisingly, we can replace in the T4 genome the small subunit gene 16 with genes encoding much larger (18 to 45 kDa) gp16-GFP and gp16-mCherry fluorescent fusion proteins without loss of function. FRET measurements of two fusion gene infected bacteria show that low level expression from the T4 genome also produces small subunit fusion protein complexes comparable to the purified rings proteins. Fluorescence approaches in vivo may allow us to determine our hypothesis that double rings synapsing DNA duplexes initiate packaging is correct. [1] Dixit et al, PNAS, 2012 [2] Dixit et al, Virology, 2013 [3] Sun et al, Cell, 2008

[4] Oram et al, JMB, 2008 [5] Itsathitphaisarn et al, Cell, 2012 [6] Lin et al, JBC, 1997.

41

Mechanism of DNA packaging initiation by the small and large terminase proteins of bacteriophage T4

S. Gao1, T. I. Alam1, B. Draper1 and V. B. Rao1

1 The Catholic University of America, 620 Michigan Ave. NE, Washington, DC 20064, USA

In tailed bacteriophages, an empty procapsid is filled with viral genome by a DNA packaging machine situated at a special fivefold portal vertex. The packaging machine consists of a “small terminase” and a “large terminase”. At the initiation of packaging, the small terminase binds to the viral genome, recruits the large terminase that cuts the DNA, and the complex docks on the portal and inserts the free DNA end into the translocation channel. The stoichiometry of the terminase complex and the mechanism of packaging initiation are poorly understood.

The small terminases consist of three domains: an N-terminal domain containing a putative DNA binding helix-turn-helix motif, a central coiled coil motif that oligomerizes into rings of 8- to 12-mers with a central channel that can accommodate dsDNA, and a C-terminal β-barrel domain that binds to the large terminase protein. To dissect the genome binding mechanism by the domains, we established an in vitro gel shift assay to demonstrated binding of DNA to the phage T4 small terminase, gp16. The binding results of domain-truncation constructs suggested that both the N- and C-terminal domains might be important. The central channel doesn’t appear to be essential, as mutation of four positively charged amino acids present in the channel, or partial/complete deletion of the 2nd central coiled coil helix, did not disrupt DNA binding of gp16 or plaque forming ability of the mutant phage. The data support a gp16-DNA binding model in which DNA wraps around the periphery of gp16 oligomer.

The T4 large terminase, gp17, has two separate DNA binding sites in the C-terminal domain, which are dedicated for DNA cleavage and translocation, respectively. By mass spectroscopy, we identified a third potential DNA binding site that links these two sites to form a continuous DNA binding surface. Mutants of the third site have significantly reduced DNA binding and nuclease activity and cannot package DNA. Structural modeling shows that the surface formed by the three sites can accommodate a DNA molecule with a bend angle of ~60˚, reminiscent of λ IHF and E. coli Hu proteins. Indeed, DNA binding assays show that gp17 binds up to 10 times more “bent” DNA than “straight” DNA. Mass spectroscopy analysis of a high affinity DNA binding mutant holding a ~ 30 bp DNA fragment confirmed that this DNA binding surface spans all the three sites.

Taken together, we propose a packaging initiation model in which binding of the genome DNA to gp16 in a wrapping-around manner presents a bent DNA substrate to gp17, and the bent surface wraps through gp17’s nuclease and translocation binding sites. Binding of DNA to both sites simultaneously allows gp17 to generate a single cut at the nuclease site and release the free end into the translocation channel without repositioning the DNA or reorienting the complex.

42

Deep sequencing the RNA encapsidated by viruses: mapping

of RNA recombination; discovery of functional motifs; and

packaging of host RNAs including retrotransposons

A. Routh1, T. Domitrovic1, P. Ordoukhanian1, J.E Johnson1

1 The Scripps Research Institute, La Jolla, CA

RNA recombination within viral genomes is a powerful driving force behind the evolution and adaption of RNA viruses and has been attributed to be the source of outbreaks of new virus strains including Echovirus and Poliovirus as well as the emergence of entirely new viruses such as SARS-CoV.

Using Deep Sequencing (RNAseq), we have devised a method to map viral RNA recombination events with single-nucleotide precision. We have applied this to analyse the genomic polymorphism and quasi-species present within an animal +ssRNA non-enveloped virus, Flock House Virus (FHV). This revealed prolific RNA recombination providing a highly sensitive and quantitative description of the complex mutational landscape of the transmissible viral genome. Recombination events that remove functional motifs are negatively selected. Consequently, the distribution of recombination events reveals the genomic motifs required for viral replication and encapsidation and so functional viral motifs can be discovered de novo without any prior knowledge of the viral lifecycle.

In addition to the viral genome we found that host mRNAs, rRNA, non-coding RNAs and retrotransposons are readily packaged. The packaging of these host RNAs elicits the possibility of horizontal gene transfer between eukaryotic hosts that share a viral pathogen.

43

Inhibition of Primary Translation of the Viral Genome is the Dominant Mechanism of Super-Infection Exclusion in Sindbis

Virus

O. Azizgolshani1, C. M. Knobler1, and W. M. Gelbart1

1Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Dr. East, Los Angeles, CA 90095

The phenomenon of super-infection exclusion (or homologous interference) has been observed for a wide variety of viruses, including Alphaviruses – a genus of enveloped plus-strand ssRNA viruses. The defining characteristic of homologous interference is the inhibiting or impeding of viral growth in cells that have been previously infected by the same or a homologous virus. Various mechanisms for this interference have been proposed, including inhibition of entry [1], of disassembly [1], and of genome replication [2]. Protein synthesis from RNA genes occurs at two time-displaced points in the viral life cycle: first, the nonstructural genes coding for the RNA-dependent RNA polymerase are translated, whose action on the viral genome results in the synthesis of minus-strand genome copies and subsequently of plus-strand copies of the full-length RNA and the subgenomic mRNA. Second, the structural proteins (capsid and membrane proteins) are translated from the subgenomic mRNA. Essentially, all the amplification steps in the viral life cycle depend on the successful primary translation and availability of many copies of nonstructural proteins. Here we examine the conditions that establish the super-infection inhibition effect for Sindbis virus and investigate the mechanism of interference by a quantitative approach. We show that the primary translation of super-infecting (or super-transfecting) viral genome is inhibited progressively as the duration of the resident infection – prior to introduction of the super-infecting virus – is increased. As the downstream processes (e.g. structural protein synthesis) demonstrate a similar decline profile, we identify translation inhibition as the main mechanism for the super-infection exclusion phenomenon in Sindbis virus. [1] Singh, I. R., et al. Virology 231, 59-71 (1997). [2] Stollar, V. and T. E. Shenk, J. Virol. 11, 592-595 (1973).

44

Unravelling the tangled processes of RNA Replication and

Encapsidation in Cowpea mosaic virus

Pooja Saxena1, Yulia Meshcheriakova1, Keith Saunders1 and

George Lomonossoff1

1 Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, UK

A fundamental question in virology is how viruses package their genomes to form stable virions. For a number of plant and animal RNA viruses, it has been established that the processes of RNA replication and RNA packaging are coupled. However, not much is known about ‘how’ these two processes are linked to each other. The link could be recognition of a signal encoded within the RNA or binding of the replicating RNA to a protein within the replication complex. To answer these questions, we investigated functional coupling of replication and encapsidation in a plant virus of the Comoviridae family, Cowpea mosaic virus.

Cowpea mosaic virus (CPMV) has a genome comprising two single-stranded positive-sense RNA, separately encapsidated in icosahedral particles of about 30 nm in diameter. When only the coat proteins are present in a host cell, empty (RNA-free) CPMV virus-like particles are generated in plants [1]. So, how is it that the virus capsid only packages wild-type RNA and not any other host RNA including the mRNA encoding viral sequences?

To study the processes of virus replication and encapsidation in CPMV, several replication-deficient mutants of RNA-1 and RNA-2 were created. Analysis of particles generated upon expression of various combinations of mutant RNAs suggests that active replication of RNA is needed for encapsidation. In addition to understanding two fundamental processes in the viral lifecycle, the long-term goal of this work is to develop a system to package ‘RNA of choice’ in CPMV capsids. The capsid can then be chemically modified for targeted delivery of the packaged nucleic acid [2].

[1] Saunders, K., Sainsbury, F. & Lomonossoff, G. P. Virology 393(2):329-37 (2009). [2] Steinmetz, N. F., Lin, T., Lomonossoff, G.P. & Johnson, J.E. (2009) Current Topics in Microbiology

and Immunology 327:23-58 (2009)

45

Regulating the switch between (ds) replicative form and (ss)

genomic DNA biosynthesis: old adversaries and new genes.

S. Doore1 and B. Fane1

1 The Bio5 Institute, University of Arizona, Tucson, AZ 58721,USA

During øX174 DNA replication, protein C mediates the switch between double-stranded (ds) and single-stranded (ss) DNA biosynthesis. Although the molecular mechanism has not been widely studied, Aoyama et al. [1] speculated that it involves two antagonists: viral protein C and the host cell, ssDNA binding protein ssB. In this model, ssB, which is required for ds DNA biosynthesis, is displaced by protein C, which promotes ss DNA synthesis. øX174 and α3 growth was examined in cells over-expressing cloned C or ssb genes. While the cloned gene induction affected both species, the effects on α3 were considerably more pronounced. Hence, it was used in further studies. The cloned α3 C gene complemented an α3 nullC mutant, but inhibited wild-type plaque formation, indicative of a dosage dependent phenomenon. Viral DNA replication intermediates were isolated and characterized from wild-type infected cells. Consistent with the model, cloned C gene induction decreased ds DNA levels. Mutants resistant to cloned C gene expression were isolated. They alter the origin of DNA replication, indicating protein C and ssB directly compete at binding the origin. In cells expressing the cloned ssB gene, ds DNA synthesis was not affect, whereas ssDNA synthesis was significantly reduced. Mutations conferring resistance to cloned ssB gene expression were located upstream of gene C. The mutations produce stop codons in frame with the downstream C gene. The results of subsequent genetic analyses strongly suggest that this 5’ extended ORF is translated from a rare, upstream, alternate start codon. This gene arrangement is conserved in all α3-like microviruses, but not found in the closely related øX174-like phages, which contain only one detectable C gene. In α3, both C genes must be eliminated in the phage to produce a nonviable nullC phenotype. Clones of either gene complemented nullC mutants, but only the smaller version strongly inhibited wild-type plaque formation. Thus, experimental results support the antagonistic protein model, provide additional mechanistic details, and elucidate an active evolutionary node of microvirus divergence.

[1] A. Aoyama, R. K. Hamatake, R. Mukai, and M. Hayashi J. Biol. Chem 258, 5798-5803 (1983).

46

Particle morphogenesis: the major barrier to horizontal gene exchange between Microviruses

Sarah M. Doore1 and Bentley A. Fane1

1 The Bio5 Institute, University of Arizona, Tucson, AZ 85721 USA

The single-stranded DNA microviruses fall into three clades, represented by φX174, G4, and α3 [1]. Their atomic structures are nearly superimposable, and interactions between structural proteins are highly conserved [2,3]. Thus, microviruses are ideal for studying the consequences of horizontal gene exchange on the protein level. Genetically chimeric virions were constructed by exchanging the major spike (G) genes between φX174 and G4. While there is a large degree of natural variation among the G4-like phages, variation within the φX174 clade is minimal. Therefore, genes were exchanged into backgrounds that appear to tolerate different degrees of natural variation. The G4-φXG chimera was viable only below 33°C. At restrictive temperatures, morphogenesis was blocked early in the assembly pathway, at the step mediated by the internal scaffolding protein. Mutations that suppressed this temperature-sensitive assembly defect were selected and characterized. All suppressors involved internal scaffolding protein function. The phenotype of the φX-G4G chimera was much more complex. This chimera required exogenous expression of two wild-type φX174 genes, encoding the major spike (G) and the minor spike (H) proteins. This observation suggests that the inhibitory barrier involves non-productive protein interactions between G4 G and φX174 H. A viable chimera was ultimately recovered: however, a series of targeted, sequential selections were necessary. The first selection was required to overcome an inhibitory interaction between the newly exchanged major spike protein G and the indigenous φX174 minor spike protein H. This involved changes in the C-terminal encoding region of the G4 G gene. The next selection allowed productive incorporation of the foreign major spike protein, though exogenous expression of the gene was still required. This involved mutations at sites governing coat-external scaffolding protein interactions and packaging. A mutation at a terminator appears to increase overall protein expression of structural genes: this may indicate that an increase in protein concentrations is necessary for productive assembly. Finally, a selection for viability without exogenous expression of any major spike gene was conducted. The mutations recovered from this selection again appear to involve external scaffolding protein interactions and elevations in intracellular structural protein levels. These data indicate that the major species barrier involves particle morphogenesis, which can be made more thermodynamically favorable by altering scaffolding protein interactions and elevating the concentration of the reactants during assembly. [1] Rokyta D.R. et al. J. Bacteriol. 188, 1134 (2006) [2] McKenna R. et al. J. Mol. Bio. 256, 736 (1996) [3] Bernal R.A. et al. J. Mol. Bio. 325, 11 (2003)

47

Using a Hamiltonian paths approach to analyze the RNA organization observed in cryo-electron tomograms of pilus-

bound MS2

J.A. Geraets1, K.C. Dent2, E.C. Dykeman1, N.A. Ranson2, P.G. Stockley2

and R. Twarock1 1 York Centre for Complex Systems Analysis, University of York, United Kingdom 2 Astbury Centre for Structural Molecular Biology, University of Leeds, United Kingdom

Assembly of single-stranded (ss) RNA viruses involves packaging an inherently asymmetric RNA molecule into a highly symmetric container formed from multiple copies of either a single coat protein subunit or several quasi-equivalent ones. Cryo-electron microscopy of RNA phages from the Leviviradae shows that the genomic RNA forms two highly ordered shells, with the outer forming a cage structure that is in direct contact with the capsid protein layer, that presents a symmetric lattice of RNA binding sites. However, such structures are icosahedrally averaged and do not reveal the molecular details of how the cage structure is realized by a single RNA molecule, or whether the conformation of the genome is the same in each copy of the virus. Previously, using a concept from graph theory called Hamiltonian paths, we enumerated all possible ways in which a genomic RNA could be organized within such a virus particle [1]. Each such configuration corresponds to an order of addition of coat proteins to a growing capsid, and hence to a different assembly pathway. Hamiltonian paths therefore also define all possible assembly pathways of the virus. Recently, an asymmetric structure of the RNA phage MS2, bound to its natural receptor has been determined [2], which suggests that the viral genome may adopt a specific tertiary structure in the infectious particle. However, the limited resolution of the current structure means that it is not possible to understand the organization of the entire RNA molecule from these data alone. We have developed an interdisciplinary method to compare the density in the tomographic data with the list of all possible Hamiltonian paths, which gives additional insights into the organization of the outer RNA shell, and thus into the structure and function of the infectious virus.

[1] E.C. Dykeman, P.G. Stockley, R. Twarock. J. Mol. Biol. Forthcoming (2013)

doi:10.1016/j.jmb.2013.06.005. [2] K.C. Dent, R. Thompson, A.M. Barker, J.A. Hiscox, J.N. Barr, P.G. Stockley, N.A. Ranson. Structure

21, 1225-1234 (2013).

48

Structure-function analysis of the phiX174 DNA pilot protein, prefabricated on-site tail assembly.

Aaron P. Roznowski1, Lindsey N. Young1, Lei Sun2, Michael G. Rossmann2, Bentley A. Fane1

1 The BIO5 Institute, The University of Arizona, Tucson, AZ 85721 USA 2 The Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 USA

The phiX174 DNA pilot protein H transports the ssDNA viral genome into the host cell. The piloting mechanism has long been an area of speculation. Unlike many double stranded DNA phages, phiX174 is strictly icosahedral and lacks a visible tail. During capsid assembly, pilot protein monomers are incorporated into pentameric coat protein assembly intermediates. The external scaffolding protein organizes 12 of these pentameric intermediates into a procapsid, a reaction that has been reconstituted in vitro. Once assembly is complete, each particle should contain 12 H protein monomers, presumably one at each vertex; however their exact location and structure in the completed capsid is unknown. Recently, the X-ray structure of the protein’s central domain was solved (See abstract by Sun et al.). The structure contains ten monomers oligomerized into an alpha helical barrel with dimensions consistent for the passage of the circular ssDNA genome across the cell wall. We are currently conducting genetic and biochemical analyses to test structure-based hypotheses and to elucidate the functions of the structurally unresolved N and C termini. To date, mutations in the highly hydrophobic N terminus, which is predicted to contain a transmembrane helix, affect host cell attachment. Using the atomic structure as a guide, mutations were made in the alpha helical barrel. These mutations were designed to inhibit the helix-helix interactions required for tube formation. In vivo, these proteins are incorporated into uninfectious virus like particles, but in vitro these mutant proteins do not oligomerize. This domain, like other coiled-coil structures, contains repeated heptad and hendecad motifs. A full length construct with a deleted hendecad motif was exogenously expressed. The construct fails to compliment a mutant lacking the wild type protein and inhibits wild type plaque formation. Resistance mutants have been isolated, and are being characterized, as well as the biochemical basis of inhibition. The C-terminus lacks any predicted secondary structure, but is hypothesized to interact with the major spike protein. A preliminary genetic analysis has shown that function is preserved, albeit at a lower level, when the last three residues are deleted. Mutations in the preceding 10 residues are poorly tolerated.

49

Horned cyanophage Syn5 assembly proceeds through a scaffolding and portal containing procapsid

Cameron Haase-Pettingell2, Desislava A. Raytcheva1, Preeti Gipson 3, Wei Dai3, Wah Chiu3,

Jacqueline M. Piret1, and Jonathan King2+

1 Department of Biology, Northeastern University, Boston MA 02115 2 Department of Biology, Massachusetts Institute of Technology, Cambridge MA 02139, 3 Baylor College of Medicine, Houston, TX

Syn5 is a marine cyanophage propagated under laboratory conditions on the marine photosynthetic cyano bacterial strain Synechococcus WH8109. Despite the major impact of cyanophages on life in the oceans, there is limited information on cyanophage intracellular assembly processes within their photosynthetic hosts. Cryo-electronmicroscopy reveals a long slender horn on the vertex opposite the tail complex, and a distinct pattern of outer capsid knobs on the icosahedral shell. One-step growth curve of Syn5 demonstrated a short cycle with an eclipse period of ~45 min and a latent phase of ~60 min, and with a burst size of 20–30 particles per cell at 28 ºC. The scaffolding protein of Syn5, absent from virions, was identified in the lysates and expressed from the cloned gene. The knobs appear to represent gp56, gp57, and gp58 (16 kDa). The unique horn proteins have been identified as gp53 (48 kDa) and gp54 (65 kDa) through immuno-electron microscopy. Particles lacking DNA, but containing the coat and scaffolding proteins, were purified from Syn5-infected cells using CsCl centrifugation followed by sucrose gradient centrifugation. Electron microscopic images of the purified particles showed shells lacking condensed DNA, but filled with protein density, presumably scaffolding protein. Sucrose gradients of pulse-chase labeled time points revealed the progression of coat from the soluble (top of the gradient) at an early time to a procapsid like species at a later time point. In addition, an expanded capsid was observed (mid-gradient). These findings suggest that the cyanophages form infectious virions through initial assembly of scaffolding-containing procapsids, similar to the assembly pathways for the enteric dsDNA bacteriophages. Since cyanobacteria predate the enteric bacteria, this procapsid mediated assembly pathway may have originated with the cyanophages.

50

Visualizing the Internal Structure of Bacteriophage T7 by Bubblegram Imaging.

Naiqian Cheng1, Weimin Wu1, Norman R. Watts2 and Alasdair C. Steven1

1 Laboratory of Structural Biology Research 2 Protein Expression Laboratory, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda MD 20892, U.S.A.

In mature T7 virions, as in many other bacteriophages, the genome is wrapped around the portal axis in layered coaxial coils whose degree of ordering decreases towards the center of the particle [1]. T7 also houses a cylindrical protein assembly called the core, mounted on the interior surface of the portal vertex [2]. In infection, the core is dismantled and injected into the host bacterium [3], along with the genome. How these two injection processes are coordinated has not been clear. We have investigated them by a visualization technique called bubblegram imaging [4], which exploits the fact that in the final stage of radiation damage by cryo-electron microscopy, protein molecules explode, generating bubbles of hydrogen gas that mark their locations. Proteins embedded in DNA bubble earlier than free-standing proteins and DNA does not bubble under the same conditions. A 3D reconstruction from fifth-exposure images (~ 85 electrons/Å2) depicts a bipartite cylindrical gas cloud in the core region. In the portal-proximal half of the cloud, the axial region, which was initially occupied by dense material, is gaseous whereas in the portal-distal half, the axial region is occupied by a dense rod. This suggests that they respectively represent (part of) core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles also appear at other (non-core) internal sites and remain smaller than core-derived bubbles. They appear to mark the locations of minor internal proteins, perhaps residual (non-expelled) scaffolding protein. [1] E. Cerritelli et al. (1997) Cell 91:271-280. [2] P. Serwer (1976) J. Mol. Biol. 10:271-291. [3] B. Hu, W. Margolin, I.J. Molineux & J. Liu (2013) Science 339:576-579. [4] W. Wu, J. A. Thomas, N. Cheng, L. W. Black & A. C. Steven (2012) Science 335:182.

51

Why do pauses happen during bacteriophage DNA ejection?

M. de Frutos1, A. Leforestier2 and F. Livolant2

1 Institut de Biochimie et Biophysique Moléculaire et Cellulaire, CNRS UMR 8619; Bât430, Centre d'Orsay, 91405 Orsay Cedex, France. 2 Laboratoire de Physique des Solides, Bât 510, Univversité Paris Sud, 91405 Orsay Cedex, France

It is possible to follow the ejection of DNA from the bacteriophage in vitro after interaction of the phage with its purified receptor. Experiments have been conducted with T5, Lambda and Spp1 by using different methods of observation and multiple protocols. Fluorescence microscopy let us visualize in real time the ejected part of the DNA and measure ejection speed [1-3]. CryoTEM experiments let us visualize the portion of DNA kept inside the capsid at different steps of the ejection and provides information on its structure [4]. Experimental requirements and constraints are different but both approaches reveal the presence of pauses during T5 ejection (moments where the ejection speed slows down to zero) allowing partially filled capsids to be imaged. We propose to compare data collected by these two methods and to discuss the importance of ionic conditions and presence/absence of a support film in the location of these pauses (article in preparation). Interesting comparisons can be done with simulation approaches [5,6]. We will then discuss the reasons why pauses have not been detected in other phages like Lambda.

[1] S. Mangenot, M. Hochrein, J. Radler, L. Letellier, Current Biology 15, 430 (2005) [2] P. Grayson, L. Han, T. Winther, R. Phillips, Proc. Natl. Acad. Sci. 104, 14652 (2007) [3] N. Chiaruttini, M. de Frutos, E. Augarde, P. Boulanger, L. Letellier, V. Viasnoff, 99 , 447 (2010) [4] A. Leforestier, F. Livolant, J. Mol. Biol. 396, 384 (2010) [5] A. S. Petrov and S. C. Harvey, Structure15, 21 (2007) [6] I. Ali, D. Marenduzzo, J. Chem. Phys. 135, 095101 (2011)

52

Conserved infection mechanism of bacterial viruses: ΦX174

forms a tail for DNA transport

L. Sun1, L. N. Young2, S. P. Boudko1, A. Fokine1, X. Zhang1, E. Zbornik1,

M. G. Rossmann1, B. A. Fane2

1 Purdue University, West Lafayette, IN, USA 2 University of Arizona, Tucson, AZ, USA

Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls: barriers that can contain two lipid bilayers and a peptidoglycan layer [1]. Many bacteriophages utilize a tail to perform this function, whereas tailless phages rely on host organelles, such as plasmid-encoded receptor complexes and pili [2-5]. However, the tailless, icosahedral, single-stranded (ss) DNA ΦX174-like coliphages do not fall into these well-defined infection paradigms. For these phages DNA delivery requires a DNA pilot protein [6]. Here we show that the ΦX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host’s periplasmic space to the cytoplasm. The 2.4 Å resolution crystal structure of the in vitro assembled H protein’s central domain consists of a 170 Å-long α-helical barrel. The tube is constructed of 10 α-helices with their N-termini arrayed in a right-handed super-helical coiled-coil and their C-termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrated that the tube is essential for infectivity but does not affect in vivo virus assembly. Both ends of the H protein contain potential trans-membrane domains, which could anchor the assembled structure into the inner and outer cell membranes. Thus, these tubes could span the periplasmic space or cell wall adhesion patches to transport the viral genome into the host’s cytoplasm. The central channel of the H protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host. [1] Silhavy, T. J., Kahne, D. & Walker, S. Cold Spring Harb. Perspect. Biol. 2, a000414,

doi:10.1101/cshperspect.a000414 (2010). [2] Molineux, I. J. & Panja, D. Nat. Rev. Microbiol. 11, 194-204 (2013). [3] Van Duin, J. & Tsareva, N. in The Bacteriophages (ed R. Calendar), pp. 175-196 (Oxford Press,

2006). [4] Russel, M. & Model, P. in The Bacteriophages (ed R. Calendar), pp. 146-160 (Oxford Press, 2006). [5] Grahn, A. M., Butcher, S. J., Bamford, J. K. H. & Bamford, D. H. in The Bacteriophages (ed R.

Calendar), pp. 161-170 (Oxford Press, 2006). [6] Jazwinski, S. M., Lindberg, A. A. & Kornberg, A. Virology 66, 283-293 (1975).

53

A pH-dependent conformational switch in the tail needle gp26 triggers genome ejection

A. Bhardwaj1, D.J. Taylor2, S.R. Casjens3, G. Cingolani1

1 Thomas Jefferson University, Dept. of Biochemistry and Mol Biology, 233 S. 10th street, Philadelphia, PA 19107. USA 2 Case Western Reserve University, Department of Pharmacology, School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106, USA 3 University of Utah School of Medicine, Department of Pathology, Salt Lake City, UT, 84112, USA

Members of the Podoviridae family encode a specialized tail needle fiber essential for genome stabilization and host cell-surface penetration [1]. In P22, this tail needle (gp26) forms a ~240Å long trimeric coiled coil fiber, only 25-35Å in diameter [2]. The needle is inserted into the portal vertex structure at the end of packaging to stabilize the highly condensed genome inside the capsid [3]. During infection, gp26 is released from virions into Salmonella enterica suggesting a role in genome ejection [4]. The N-terminal plugging-tip of gp26 (residues 1-60) binds to tail factor gp10 and forms the plug that closes P22 portal channel after packaging [5]; this region is extraordinarily well conserved in hundreds of P22-like phage and prophages, suggesting a conserved function. We have determined crystal structures of P22 and HK620 tail needles at acidic and neutral pH. These structures reveal that the N-terminal plugging-tip of gp26 is unstructured at neutral pH, whereas it folds largely into a trimeric helical bundle at acidic pH. Accordingly, gp26 is protease sensitive and less stable in solution at neutral pH than at pH 5.0, suggesting a pH-dependent unfolding of its N-termini. Direct observation of gp26 fibers by electron microscopy suggests a pH-dependent shortening of the fiber N-termini, consistent with crystallographic data. Likewise, a reanalysis of high-resolution asymmetric cryo-electron microscopic reconstructions of mature phage P22 reveals that the conformation of gp26 visible in these reconstructions is consistent with the neutral pH-conformation, which has unstructured N-termini. We propose that the tail needle adopts a pre- and post-ejection conformation. The post-ejection conformation, which presents a folded N-terminal tip and is stabilized by acidic pH, commits the fiber to be injected through the periplasm, avoiding rebinding to the portal vertex. [1] A. Bhardwaj, N. Walker-Kopp, S.R. Casjens, G. Cingolani. J. Mol. Biol. 391(1), 227 (2009) [2] S.A. Olia, S.R. Casjens, G. Cingolani. Nat. Struct. Mol. Biol. 14(12), 1221 (2007) [3] H. Strauss, J. King. J. Mol. Biol. 172, 523 (1984) [4] V. Israel. J. Virol. 23, 91 (1977) [5] A. Bhardwaj, S.A. Olia, N. Walker-Kopp, G. Cingolani, J. Mol. Biol. 371(2), 374 (2008)

54

Structural insights into the bacteriophage Φ29 dsDNA packaging motor

Huzhang Mao1, Mitul Saha1, Emilio-Reyes Aldrete1, Paul Jardine2, Shelley Grimes2, and Marc Morais1

1 Biochemistry and Molecular Biology, Sealy Center for Structural and Molecular biology, University of Texas Medical Branch, Galveston, TX 77555. USA 2 Diagnostic and Biological Sciences and Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455. USA

The tailed dsDNA bacteriophage ø29 packages its 19.3 Kb genome into a pre-assembled prolate icosahedral procapsid using a transiently assembled phage-encoded molecular motor. This process is remarkable considering that compaction of DNA to near crystalline densities within the confined space of the capsid requires that the motor work against considerable entropic, enthalpic and DNA bending energies. The motor is powered by a homomeric ring ATPase belonging to the ancient and ubiquitous ASCE superfamily (Additional Strand Catalytic E (glutamate)). The mechanism of the DNA packaging ATPase in ø29 has been investigated using a combination of X-ray crystallography and cryo-electron microscopy. Comparison of cryo-EM structures of the motor in the apo-state, the substrate-bound state, and a DNA-translocating state suggest domain rearrangements in the ATPase that occur during genome packaging. Additionally, an asymmetric reconstruction of particles stalled during DNA packaging shows specific interactions between a single unique subunit in the ATPase ring and the translocating DNA genome, as well as interactions between adjacent subunits around the ATPase ring. Finally, cryo-EM and SAXS analysis of the packaging ATPase from the lactococcis lactis phage asccø28, a distant relative of ø29, provides insight into the universality of various features of DNA packaging motors in ø29-like phages.

55

X-ray structure of the small terminase protein from the

thermophilic bacteriophage G20C

Juan Loredo-Varela1, Maria Chechik1, Callum Smits1, Leonid Minakhin2, Konstantin Severinov2, Huw Jenkins1, Alfred Antson1

1 York Structural Biology Laboratory, University of York, York YO10 5DD, UK 2 Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, NJ 08854, USA

Small terminase (ST) plays two important roles in the assembly of dsDNA phages, first by recognising the viral DNA and secondly by modulating the activity of the large terminase [1]. X-ray structures are already available for small terminase oligomers of several bacteriophages including SF6, Sf6, 44RR (T4-like) and P22 [2-5]. All have similar overall architecture and share similarities in the fold of their oligomerisation domain. However, there are differences in the fold of the DNA-binding domain. So far no structural information was available for small terminases from thermophilic phages. We determined the X-ray structure of a small terminase protein from the thermophilic bacteriophage G20C at 2.5Å resolution [6]. There are 9 subunits per oligomer, consistent with solution data obtained by SEC-MALLS. The putative DNA-binding N-terminal domain is composed of three α-helices and is connected to the central oligomerisation domain by a short linker. The C-terminal segment of the protein contains a pair of long antiparallel α-helices linked by a short β-hairpin. 9 pairs of such helices form the central oligomerisation domain which contains a tunnel with a radius of ~22 Å. Experiments to probe interaction of the small terminase protein with DNA are now in progress. [1] Casjens, S.R. Nat Rev Micro. 9, 647-657. (2011) [2] Buttner, C.R., M. Chechik, M. Ortiz-Lombardia, C. Smits, I.O. Ebong, V. Chechik, G. Jeschke, E. Dykeman, S. Benini, C.V. Robinson, J.C. Alonso and A.A. Antson. P Natl Acad Sci USA. 109, 811-816. (2012) [3] Zhao, H., C.J. Finch, R.D. Sequeira, B.A. Johnson, J.E. Johnson, S.R. Casjens and L. Tang. P Natl Acad Sci USA. 107, 1971-1976. (2010) [4] Sun, S., S. Gao, K. Kondabagil, Y. Xiang, M.G. Rossmann and V.B. Rao. P Natl Acad Sci USA. 109, 817-822. (2012) [5] Roy, A., A. Bhardwaj, P. Datta, Gabriel C. Lander and G. Cingolani. Structure. 20, 1403-1413. (2012) [6] Minakhin, L., M. Goel, Z. Berdygulova, E. Ramanculov, L. Florens, G. Glazko, V.N. Karamychev, A.I. Slesarev, S.A. Kozyavkin, I. Khromov, H.-W. Ackermann, M. Washburn, A. Mushegian and K. Severinov. J. Mol. Biol. 378, 468-480. (2008)

56

Role of the Adenovirus Core Protein VII in Virus Assembly,

Genome Packaging and Gene Expression

Philomena Ostapchuk1 and Patrick Hearing1

1 Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11794.

The Adenovirus (Ad) linear double stranded DNA genome has the unusual property in the virus world of being tightly associated with a virus-encoded, protamine/histone-like protein (core protein VII) in the viral capsid. In addition toiprotein VII, Ad proteins mu and V are also associated with the DNA in the virion. This core structure has been difficult to image by cryoEM, apparently lacking symmetry, and little is known about the structure of core. Cores can be isolated from the external capsid, and VII has the property of condensing the DNA. Isolation of Ad DNA/VII complexes from virions show a “bead on a string” appearance in EM. Furthermore, upon infection, protein VII stays associated with Ad DNA in the nucleus during the early phase of the life cycle. This DNA/protein complex presents an interesting conundrum with regard to viral DNA packaging. It is hypothesized that Ad packages its DNA in a fashion similar to the dsDNA bacteriophage paradigm; that is, the DNA is inserted into a pre-formed empty capsid using a packaging motor, likely the Ad IVa2 protein. However, it is not know whether the DNA genome is packaged as naked DNA, followed by the insertion of the core proteins, or as a DNA/protein complex. Either way, the motor would be required to recognize and facilitate the movement of a heterogenous structure(s). We constructed an Ad that conditionally expresses the VII protein to explore its role in packaging of the Ad genome. Our results demonstrate that contrary to popular belief, protein VII is not required for Ad genome packaging into the virion. Virus particles containing the Ad genome, but lacking protein VII, can be efficiently isolated. The VII-less virus particles, however, are not infectious even though the genomes can be delivered to the cell nucleus. Furthermore, it appears that the VII-less genomes are not templates for transcription.

57

Coordination and Communication in a Complex Biological Nanomotor

P. J. Jardine1, Bora Kuyomba1, Rockney Atz1, and Shelley Grimes1

1 Department of Diagnostic and Biological Sciences, University of Minnesota, 18-242 Moos Tower, 515 Delaware St. SE, Minneapolis, MN, USA

It has been recently determined that the DNA translocating mechanism of the Bacillus subtilis phage ø29 is incredibly complex. Rather than a simple mechanism whereby individual subunits in the oligomeric ATPase take turns moving a small allotment of DNA into the head, it appears that ø29 has evolved a mechanism whereby all subunits in the motor participate as a group in a highly coordinated mechanochemical cycle [1,2]. Briefly, the DNA translocation cycle is proposed to involve the ordered turnover of spent nucleotide and binding of ATP by all of the motor ATPase subunits during a static “dwell” followed by a rapid, ordered hydrolysis of the full ATP complement coupled to DNA movement during the translocating “burst”. In order to achieve the level of coordination demanded by such a mechanism, subunits within the motor must exchange information regarding their nucleotide-bound state. Here we present a preliminary investigation of the nature of communication within the ø29 packaging motor, focusing primarily on the inherent communication within the ATPase ring. Using mutagenesis and bulk biochemical assays we demonstrate the role of trans-acting motor components and present the network of motor communication developed thus far.

[1] G. Chistol, et al., Cell 151(5):1017-1028 (2012). [2] J. Moffitt, et al., Nature 457:446-450 (2009)

58

A phage-encoded HNH protein is required for HK97 terminase function

Smriti Kala1, Paul D. Sadowski2, Alan R. Davidson2,3, and Karen L. Maxwell1,2

1 Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada, M5S 3E1 2 Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada, M5S 1A8 3 Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8

Terminase enzymes are an essential component of the viral assembly process for all tailed phages and Herpes viruses. The genome packaging reaction of HK97 has not been directly examined, but the conservation of morphogenetic genes and the mechanism of DNA-packaging amongst tailed phages suggests that DNA packaging in HK97 will be similar to previously characterized phages such as l. In addition to the terminase enzyme and portal protein, the l packaging reaction requires the activity of the phage-encoded molecular chaperone, gpFI, in vivo. While HK97 does not possess a gpFI-like packaging chaperone, the HK97 genome and a large number of other Caudovirales genomes encode an HNH family protein located adjacent to the terminase genes. HNH family proteins are widely distributed and have been found to bind to nucleic acids and often possess endonuclease activity. They are found in a variety of proteins, including site-specific homing endonucleases, colicins, S-pyocins, and restriction enzymes. We discovered that the activity of the HK97 HNH protein, gp74, is required for phage propagation in vivo. Electron microscopy of lysates produced by a phage lacking gp74 revealed an abundance of proheads and no fully assembled phages. This phenotype was identical to that of a phage lacking the large terminase subunit. In vitro cos cleavage reactions revealed that the presence of gp74 markedly stimulated terminase-mediated cleavage of the cos site. Our results imply that HNH proteins are commonly involved in terminase function in a wide variety of tailed phages.

59

Viral genome structures are optimal for capsid assembly

J.D. Perlmutter1, M.F. Hagan1

1 Martin A Fisher School of Physics, Brandeis University, 415 South St. Waltham, MA 02453 USA For many viruses, the spontaneous assembly of a capsid shell around the nucleic acid (NA) genome is an essential step in the viral life cycle. Understanding how assembly depends on the structure of the capsid proteins and the NA could promote biomedical efforts to block viral propagation and guide the reengineering of capsids for gene therapy applications. We present a coarse-grained model of capsid proteins and NAs with which we investigate assembly dynamics and thermodynamics. In contrast to recent theoretical models, we find that capsids spontaneously ‘overcharge’; that is, the NA length which is kinetically and thermodynamically optimal possesses a negative charge greater than the positive charge of the capsid. When applied to specific virus capsids, the calculated optimal NA lengths closely correspond to the natural viral genome lengths. Simulations using linear polyelectrolytes rather than base-paired NAs result in optimal lengths shorter than the viral genomes, demonstrating the importance of NA structure to capsid assembly. These results suggest that the features included in this model (i.e. electrostatics, excluded volume, and NA tertiary structure) are sufficient to predict assembly thermodynamics and that the ability of viruses to selectively encapsidate their genomic NAs can be explained, at least in part, on a thermodynamic basis [1].

[1] J.D. Perlmutter. C. Qiao, M.F. Hagan. eLife 2, e00632 (2013).

60

Mechanical Stability and Reversible Fracture of Vault

Particles

A. Llauró1, P. Guerra2, N. Irigoyen3, J. F. Rodríguez4, N. Verdaguer2, and P.J. de Pablo1

1Departamento de Física de la Materia Condensada, UAM, Francisco Tomás y Valiente 7, 28049-Madrid, Spain. 2 Institut de Biologia Molecular de Barcelona, CSIC. Baldiri I Reixac 10, 08028-Barcelona, Spain 3 Division of Virology, Department of Pathology, University of Cambridge, Tennis Court, Cambridge CB2 1QP, United Kingdom 4 Centro Nacional de Biotecnlolgía, CSIC, Calle Darwin nº3, 28049-Madrid, Spain.

Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as drug delivery containers. In this paper we study the local stiffness of individual vaults and probe their structural stability with Atomic Force Microscopy (AFM) under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we provoke single protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution AFM topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which may enable these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.

61

Segment Self-Repulsion is the Major Driving Force of Influenza Genome Packaging.

S. V. Venev1 and K. B. Zeldovich1

1 Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA The genome of influenza A virus consists of eight separate RNA segments, which are selectively packaged into virions prior to virus budding. The microscopic mechanism of highly selective packaging involves molecular interactions between packaging signals in the genome segments and remains poorly understood. In this work [1] we propose that the condition of proper packaging can be formulated as a large gap between RNA-RNA interaction energies in the viable virion with eight unique segments and in improperly packed assemblages lacking the complete genome. We then demonstrate that selective packaging of eight unique segments into an infective influenza virion can be achieved by self-repulsion of identical segments at the virion assembly stage, rather than by previously hypothesized intricate molecular recognition of particular segments. Using Monte Carlo simulations to maximize the energy gap, without any other assumptions, we generated model eight-segment virions, which all display specific packaging, strong self-repulsion of the segments, and reassortment patterns similar to natural influenza. The model provides a biophysical foundation of influenza genome packaging and reassortment and serves as an important step towards robust sequence-driven prediction of reassortment patterns of the influenza virus. The proposed self-repulsion model is directly supported by an independent in vitro study [2], which demonstrated preferential pairing between various viral RNA segments, along with the absence of homodimers (pairs of identical segments) throughout the experiment. The observed pattern of preferential pairing between segments does not match the patterns previously described in a number of cryo-EM studies [3,4], suggesting that self-repulsion (lack of self-attraction) between the segments may be an important driving force for budding of viable virions with eight unique segments. [1] S. V. Venev and K. B. Zeldovich. Phys. Rev. Lett. 110, 098104 (2013). [2] C. Gavazzi, C. Isel, E. Fournier, V. Moules, A. Cavalier, D. Thomas, B. Lina and R. Marquet. Nucl.

Acids Res. 41 (2), 1241-1254 (2013). [3] E. Fournier, V. Moules, B. Essere, J.-C. Paillart, J.-D. Sirbat, C. Isel, A. Cavalier, J.-P. Rolland, D.

Thomas, B. Lina and R. Marquet. Nucl. Acids Res. 40 (5), 2197-2209 (2012). [4] T. Noda, Y. Sugita, K. Aoyama, A. Hirase, E. Kawakami, A. Miyazawa, H. Sagara and Y. Kawaoka.

Nat. Commun. 3, 639 (2012)

62

Predicting functional units in viral capsids from quasi-rigid

capsid subdivisions

G.Polles1, G. Indelicato2, R. Potestio3, P. Cermelli4, R. Twarock2, C. Micheletti1

1 International School for Advanced Studies (SISSA), Trieste, Italy

2 York Centre for Complex Systems Analysis, Department of Mathematics, University of York (United Kingdom)

3 3 Max-Planck-Institut fur Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany

4 Dipartimento di Matematica, Università di Torino, Torino, Italy

Capsid assembly, disassembly, post-assembly structural changes and response to mechanical stress are important physical mechanisms which govern various steps of the life cycle of viruses. These mechanisms are best characterised and rationalized in terms of the typically multimeric protein units that articulate the capsid conformational mechanics or that act as basic assembly/disassembly units. We addressed the problem of identifying these fundamental blocks by developing and applying an efficient and transferable computational strategy which allows to pinpoint the basic mechanical units of a viral capsid. The method relies on elastic network models and an optimal quasi-rigid decomposition strategy. We first validated the domain-decomposition strategy against a set of well-characterized viruses (CCMV, MS2, STNV, STMV) and next formulated verifiable prediction for other viral particles such as L-A virus, Pariacoto and Polyoma, for which the basic functional units are either still unknown or debated.

63

STIV ATPase and Genome Structure

Mark Young1

1Montana State University We have recently completed the X-ray crystal structure and genetic analysis of the STIV ATPase. This analysis suggests that the ATPase forms a multimeric structure involved with viral genomic DNA packaging and or release. In addition, we have re-evaluated the nature of the packaged viral genome which suggest that permutated linear dsDNA is the packaged form, not covalently closed circular dsDNA.

64

Late Intermediates in the Assembly of Virus Capsids

E. E. Pierson1, D. Z. Keifer1, L. Selzer1, L. S. Lee1, N. C. Contino1, J. C.-Y. Wang1, A. Zlotnick1, M. F. Jarrold1

1 Department of Chemistry and Department of Molecular and Cellular Biochemistry, Indiana University, 800 E Kirkwood Ave, Bloomington, IN 47405 USA The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates are expected at the end of the assembly reaction due to closure of the icosahedron. But whether late assembly bottlenecks exist has remained unknown because it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify late intermediates in the assembly of the Hepatitis B virus T=4 capsid, a complex of 120 protein dimers. Prominent metastable intermediates corresponding to 104, 111, and 115 dimers were observed. Cryo-EM observations confirm the presence of incomplete capsids. From their stability and kinetic accessibility we have predicted plausible structures for the intermediates. The intermediate with 111 dimers is attributed to an icosahedron missing one facet, and the 104-dimer intermediate is assigned to an icosahedron missing two neighboring facets.

65

Assembly of single coat proteins of filamentous phage in the membrane of Escherichia coli

Andreas Kuhn1, Martin Ploss 1 and Christian Klenner1

1 Institute of Microbiology and Molecular Biology, University of Hohenheim, 70599 Stuttgart, Germany

Filamentous bacteriophage M13 is assembled during a secretion process in the cytoplasmic membrane of E. coli. Membrane inserted phage proteins contact the single stranded phage DNA in a helical array and pass through the outer membrane within a porin-like structure composed of gp4. In the inner membrane, a complex, consisting of gp1, gp11 and thioredoxin, catalyzes the assembly process. First, membrane-inserted gp7 and gp9 proteins form a structure at the tip of the emerging phage that is then extended by a multiple array of gp8 proteins, the major coat proteins. We followed the emerging phage particles from the cell with the AFM [1]. Gp8 is synthesized as a precursor protein, termed procoat, that is membrane inserted by the YidC protein and is processed by leader peptidase.

We have studied in detail how YidC membrane-inserts the major coat proteins of M13 and Pf3, another filamentous phage. We found by cysteine disulfides that the transmembrane region of the coat proteins make close contact to YidC, 30 seconds after their synthesis [2]. Using fluorescent site-specific labeling and reconstituted YidC in proteoliposomes we were able to follow single coat proteins inserting into the membrane [3]. When the YidC protein was labeled in addition with another fluorescent dye, single FRET events could be followed in real time [4]. Analysis of these events allowed us to follow the binding of the Pf3 coat protein to YidC and its release into the membrane bilayer. The membrane traversal of an individual fluorescent dye on the coat protein takes about 2 msec.

[1] M. Ploss and A. Kuhn. Phys. Biol. 7, 045002 (2010). [2] C. Klenner and A. Kuhn A (2012) J. Biol. Chem. 287, 3769-3776 (2012). [3] S. Ernst, A-K. Schönbauer, G. Bär, M.Börsch and A. Kuhn. (2011), J. Mol. Biol. 412, 165-175 (2011). [4] S. Winterfeld, S. Ernst, M. Börsch, U. Gerken, A. Kuhn. PLOSone e59023 (2013).

66

Getting the kinks out: Unraveling the role of bacteriophage P22's spine helix during capsid maturation

Tina Motwani1 and Carolyn Teschke1

1 University of Connecticut, Storrs, CT, USA Double-stranded DNA viruses like HK97 and P22 exhibit similar modes of capsid maturation. As the capsid expands, it undergoes large scale conformational changes including thinning of the capsid shell, increase in the shell diameter, and the change in the shell morphology from round to faceted. Recent biophysical studies of HK97 have highlighted another interesting feature that occurs during the viral capsid maturation process. Johnson and colleagues have shown that in the prohead II particle, the procapsids are composed of skewed hexamers owing to the presence of a bend in the long spine helix of the coat protein. Upon capsid maturation to the head II particle, the kink in the spine helix gets straightened, thereby resulting in symmetric hexamers [1]. A similar bent “spine helix” has been observed in the cryo EM structures of P22 procapsid but not in the P22 expanded head [2] [3]. However, not much is known about the role the spine helix plays during the P22 maturation process. In addition, there is no direct evidence available to explain what triggers the kink in the spine helix to be straightened as the capsid matures. The goal of our work is to elucidate the role spine helix plays during capsid maturation. In order to achieve this, we have created single amino acid substitutions in the spine helix of P22’s coat protein. Complementation assays have shown that these single amino acid substitutions result in cold-sensitive defects. Currently, we are employing several techniques including heat-induced expansion, protease digestion and electron microscopy to explore the effects of these substitutions on the capsid maturation. [1] Gertsman, I., et al., Nature, 2009. 458(7238): p. 646-50. [2] Parent, K.N., et al., Structure. 18(3): p. 390-401 [3] Chen, D.H., et al., PNAS. 108(4): p. 1355-60.

67

Membrane-assisted virus assembly

Teresa Ruiz-Herrero 1, Michael F. Hagan2

1 Departamento de Física Teórica de la Materia Condensada, Universidad Autónoma de Madrid, Madrid, Spain 2 Martin Fisher School of Physics, Brandeis University, Waltham, Massachusetts

Enveloped viruses aquire a lipid membrane coat by budding through a host cell membrane. For many viruses assembly and budding occur simultaneously during virion formation. In this work we use computational modeling to explore how this process depends on physical parameters such as protein-membrane interactions and the domain structure within a membrane. The mechanistic principles governing assembly-driven budding, and why many enveloped viruses (including HIV and influenza) preferentially bud through membrane microdomains (e.g. lipid rafts), are poorly understood. Therefore, we have performed molecular dynamics simulations on a coarse-grained model that describes virus assembly on a fluctuating lipid membrane. Our simulations show that the membrane promotes assembly through dimensional reduction of adsorbed subunits, but also introduces barriers that inhibit complete assembly. We find an unexpected mechanism by which membrane microdomains can enhance assembly by reducing these barriers. Furthermore, the simulations demonstrate that assembly and budding depend crucially on the system dynamics via multiple timescales related to membrane deformation, protein diffusion, association, and adsorption onto the membrane. Finally, we will discuss how the predicted mechanisms can be experimentally tested.

68

The Delta Domain of Bacteriophage HK97 is required for Capsid Assembly and Portal Incorporation.

B. Oh1, R. Duda1, R. Hendrix1

1 University of Pittsburgh, 4200 Fifth Ave Pittsburgh, PA 15260. USA HK97 is an Escherichia coli bacteriophage whose capsid assembly mechanism has been studied in great detail. Despite this, little is known about the early steps of viral capsid assembly, specifically how the virus is able to make the correct sized and shaped capsid. During assembly, many viruses require α-helical scaffolding proteins to make procapsids of the proper size and shape. Bacteriophage HK97, unlike some other bacteriophages, does not possess a separate scaffolding protein, but has a 102 amino acid N-terminal extension of the major capsid protein known as the delta domain. The delta domain is believed to function as a scaffolding protein because it is required for correct assembly of the capsid, is absent in the mature virion, and is predicted to have primarily α-helical secondary structure. This domain is interesting (and challenging to study) because there is no X-ray crystallography data available, and it is only partially ordered in the cryo-EM structures of procapsids. In order to understand how the delta domain is involved in the initial assembly of the capsid shell, I use a plasmid based system to express HK97 procapsids in E. coli, and study the effects of mutations on capsid protein processing and assembly. Complete deletion of the delta domain caused the protein to precipitate and no structures were formed, suggesting that this domain is involved with protein folding, or assembly. Some point mutations cause capsid formation abnormalities resulting in tube like structures and other monsters, and when tested to see if they complement a phage with an amber mutation in the capsid gene, the mutations were unable to complement and so judged nonfunctional. These results confirm that the delta domain is involved with capsid assembly. A few mutants were able to assemble into wild-type Proheads and be cleaved by the maturation protease, but the protein produced is still nonfunctional when tested in complementation assays. A few mutations, in and surrounding a conserved region of the delta domain, are defective in portal incorporation. Changing one residue in the conserved region also abolishes protease incorporation. I also tested four deletion mutants, which delete 10, 26, 47, and 59 residues within the N terminal region of the delta domain. The shortest deletion mutant produced only uncleaved Proheads, probably because it prevents protease incorporation in the capsid. The other deletion mutants caused the protein to precipitate. Understanding how the delta domain regulates capsid assembly and how this domain may be structured will provide important details about the assembly process and the interactions that proteins make when forming macromolecular structures.

69

Norovirus capsid proteins self-assemble through biphasic kinetics via long-lived stave-like

intermediates

G. Tresset1, C. Le Cœur2, J.-F. Bryche1, M. Tatou1, M. Zeghal1, A. Charpilienne3, D. Poncet3, D. Constantin1 and S. Bressanelli3

1 Laboratoire de Physique des Solides, Université Paris-Sud, CNRS, France 2 Institut de Chimie et des Matériaux Paris-Est, Université Paris-Est, CNRS, France 3 Laboratoire de Virologie Moléculaire et Structurale, CNRS, INRA, France

The self-assembly kinetics for a Norovirus capsid protein [1] were probed by time-resolved small-angle X-ray scattering, then analyzed by singular value decomposition and global fitting. Only three species contribute to the total scattering intensities: dimers, intermediates comprising some 11 dimers, and icosahedral T = 3 capsids made up of 90 dimers. Three-dimensional reconstructions of the intermediate robustly show a stave-like shape consistent with an arrangement of two pentameric units connected by an interstitial dimer. Upon triggering of self-assembly, the biphasic kinetics consist in a fast step assembling dimers into intermediates, followed by a slow step in which intermediates interlock into capsids (see Figure). This simple kinetic model reproduces experimental data with an excellent agreement over 6 decades in time and with nanometer resolution [2]. The extracted form factors are robust against changes of experimental conditions. These findings challenge and complement currently accepted models for the assembly of Norovirus capsids.

[1] G. Tresset, V. Decouche, J.-F. Bryche, A. Charpilienne, C. Le Cœur, C. Barbier, G. Squires, M.

Zeghal, D. Poncet and S. Bressanelli, Unusual self-assembly properties of Norovirus Newbury2 virus-like particles, Arch. Biochem. Biophys., accepted for publication

[2] G. Tresset, C. Le Cœur, J.-F. Bryche, M. Tatou, M. Zeghal, A. Charpilienne, D. Poncet, D. Constantin and S. Bressanelli, Norovirus capsid proteins self-assemble through biphasic kinetics via long-lived stave-like intermediates, J. Am. Chem. Soc., accepted for publication

Figure. Kinetic scheme of Norovirus capsid assembly.

70

Capsid maturation of a non-enveloped animal virus enables subunit-specific membrane lytic activity and generates

hysteresis in structural transitions controlled by pH

T. Domitrovic1, B.M. Kearney1, T. Matsui2, J.E. Johnson1 1 The Scripps Research Institute, 10550 N. Torrey Pines Road MB-31. La Jolla, CA 92037. USA 2 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University. 2575 Sand Hill Rd, MS99, Menlo Park, CA 94025 USA Virtually, all animal viruses transition from a procapsid noninfectious state to a mature infectious state. In this work we show how the structural changes and autocatalytic reactions involved in capsid maturation can confer multi-functionality to a simple non-enveloped +ss RNA virus formed by just one kind of capsid protein. In neutral or alkaline pH, NωV capsid proteins assemble around RNA forming a round procapsid. After acidification, the capsid collapses into a compact form with distinctive quasi-equivalent contacts. This structural transition activates auto-proteolysis of the capsid protein liberating a peptide that stays non-covalently associated to the capsid. The different structural environments resulting from the quasi-equivalent subunit positions are important to specify their function. This was demonstrated by correlating the lytic activity against membranes with the generation of lytic peptides coming specifically from subunit pentamers, where they form a distinctive helical bundle pointing towards the virus surface [1]. The membrane lyses reaction occurs only in alkaline conditions, that correlate with the in-vivo environment of infection inside caterpillars mid-gut. We found that the structural transition of mature capsids back to high pH not only induces the exposure of peptides to the environment but also can induce an unexpected reconstitution of the covalent bond between the capsid protein and another subset of the peptides that were cleaved during maturation. Possible functional implications of this reaction for capsid disassembly and RNA exposure will be discussed. [1] T. Domitrovic, T. Matsui, J.E. Johnson. J. Virol. 86, 9976 (2012)

71

CryoEM of portal machinery of bacteriophage PRD1

Chuan Hong1, 2, Xiangan Liu2, Joanita Jakana2, Hanna Oksanen3, Dennis Bamford3, and Wah Chiu1, 2

1 Graduate Program in Structural and Computational Biology and Molecular Biophysics, and 2 National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, 77030 3 Department of Biosciences and Institute of Biotechnology, University of Helsinki, PO Box 56, FIN-00014 Helsinki, Finland

PRD1 is an icosahedral dsDNA bacterial virus with an inner membrane (Tectiviridae family). Based on previous X-ray crystallographic results of PRD1, its major capsid protein has similar fold as those of several other viruses such as adenovirus, PBCV-1 and STIV. However, the structure of the non-icosahedrally arranged portal complex anchored in the inner membrane remains elusive. Biochemical and immuno-electron microscopic studies have identified four proteins in the portal complex: the packaging ATPase P9, the packaging efficiency factor P6, and the integral membrane proteins P20 and P22. The goal of our study is to reveal the structures of the portal machinery of PRD1. We used single-particle electron cryo-microscopy (cryo-EM) to study the mature virion and three procapsid mutants of PRD1 using symmetry-free reconstructions. Their density maps allow us to conclude the locations and features of the four portal proteins at a unique vertex. The P20 and P22 form a hexamer of dimers embedded in the viral membrane and function as a conduit for the DNA packaging. The P20 or P22 cannot exist alone without the other. The P6 and P9 form a 12-mer of a portal complex with ATPase activity similar to other phage portal protein complex. This is the first structural evidence of the PRD1 packaging complex operating at a specific vertex, and shows the connection between the membrane and the capsid shell providing a conduit for DNA translocation in an ATPase-driven reaction. This research was supported by grants from the National Institutes of Health (P41GM103832 and R01R56AI075208), Robert Welch Foundation (Q1242) and Academy Professor Finland grants (255342 and 256518). We thank University of Helsinki for the support to EU ESFRI Instruct Centre for Virus Production and Purification used in this study.

72

Shigella Outer Membrane Proteins A & C Mediate Sf6 Genome Ejection

Kristin N. Parent1, Marcella L. Erb2, Giovanni Cardone3, Katrina Nguyen2, Eddie B. Gilcrease4,

Natalia B. Porcek1, Joseph Pogliano2, Timothy S. Baker2, 3, Sherwood R. Casjens4

1 Michigan State University, Department of Biochemistry and Molecular Biology, East Lansing, MI, 48224, USA 2 University of California, San Diego, Division of Biological Sciences, La Jolla, CA 92093, USA 3 University of California, San Diego, Department of Chemistry & Biochemistry, La Jolla, CA 92093, USA 4 University of Utah School of Medicine, Department of Pathology, Salt Lake City, UT 84112, USA

Mechanisms that control virus adsorption and DNA ejection are not well understood. Previously, bacterial outer membrane proteins (Omp) A and C were identified in lipid vesicles that co-purify with bacteriophage Sf6, implicating both of them as potential host receptors. We have determined that OmpA and OmpC mediate infection by dramatically increasing the rate and efficiency of infection. We performed a combination of in vivo studies with three omp null Shigella flexneri mutants (ompA-, ompC-, and ompA-C-), which included classic phage plaque assays, time-lapse fluorescence microscopy to monitor genome ejection at the single particle level, and cryo-electron tomography of phage attached to and “infecting” outer membrane vesicles. Lastly, we employed in vitro ejection studies that show lipo-polysaccharide and Omps are both needed for Sf6 genome release. We hypothesize that Sf6 can utilize either OmpA or OmpC for genome release and productive infection, yet OmpA is the preferred receptor.

73

Atomic model of the pyocin reveals how energy is stored to “kill”

Peng Ge1

1 UCLA

An R-type pyocin punctures a hole through a victim bacterium with a contractile assembly, killing it by dissipating its proton motif force. It shares structural and functional similarity with type six secretion systems; all contract with a sheath-tube assembly that provides energy for penetration into its target membrane. Currently, our understanding of this type of molecular machines is seriously hindered by the absence of a representative atomic structure of it, largely due to the inability of solving such structures by traditional structural biology tools. Here, by applying helical cryo-electron microscopy reconstruction, we, for the first time, show the atomic details of this contractile machine. In the loaded, pre-contraction pyocin, the two proteins, tube and sheath, are organized in coaxial helices that share a same helical symmetry and a six-fold rotational harmonics. The tube is made of stacked discs, each containing a 24-strand β-barrel, spanning six subunits, optimized for proton conduction. The sheath layer is scaffolded with the tube layer by attaching a single α-helix of each sheath subunit onto a tube subunit, electrostatic interactions between them. The energy source for contraction is a molecular battery made of sheath subunits that, upon contraction, reorganizes itself into a structure with lower electrostatic potential by better pairing the opposite charges among the subunits. These findings will allow us to better understand the aforementioned contractile systems for therapeutic purposes, either to harness a pyocin for antibacterial use or to design molecular interventions that block type six injections from a hostile bacterium.

74

The phage P22 tail needle affects DNA delivery

Sherwood R. Casjens 1

1 University of Utah

Modifications of the tip of the P22 tail needle affect DNA delivery as measured by potassium ion release.

75

Cryo-EM Single Particle Reconstruction of P2 and P7

Proteins of Cystovirus φφφφ6

Al Katz1, Garrett Katz2*, Alexandra Alimova2, Hui Wei2, Lucas Oliveria3, and Paul Gottlieb2

1 Physics Department, City College of New York, New York, NY 10031 USA 2 Sophie Davis School of Biomedical Education, City College of New York, New York, NY 10031 USA 3Graduate Center of the City University of New York, 365 Fifth Ave. New York, NY 10016, USA *Current Address: Dept. of Computer Science, University of Maryland, College Park, MD, USA

Difference maps were generated to compare high resolution single particle reconstructions of the φ6 unexpanded procapsid (PC), P2-minus (P2 protein is the RNA directed RNA-polymerase) PC, and P7-minus (P7 protein is an assembly cofactor) PC. The maps identified the location of P7 prior to RNA packaging as being at the three-fold symmetry axes. The difference maps demonstrate that in the P7-minus particles, P2 exhibits decreased order, and that P2 is less localized with reduced densities at the three-fold axes. We propose that P7 performs the mechanical function of stabilizing P2 on the inner protein P1 shell which ensures that entering viral single-stranded RNA is replicated. P7 may also help stabilize the unexpanded PC. In the complete PC, each P2 appears to be surrounded by 3 P7 proteins. P2 and P7 occupancies were estimated from density levels. In the complete PC, P7 occupancy is 57% but only 19% in the P2-minus mutant. P2 occupancy at the three-fold axis of the complete PC is 50%. P2 occupancy at the three-fold axis is only 28% in the P7-minus particles, likely the result of P2 being less localized in the P7-minus particles.

76

Gp12: a viral collagen-like protein that binds to the bacteriophage SPP1 capsid

Mohamed Zairi1, Asita Stiege2, Naima Nhiri3, Eric Jacquet3 and Paulo Tavares1

1Unité de Virologie Moléculaire et Structurale, UPR 3296 CNRS, 91190 Gif-sur-Yvette, France. 2Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, Berlin, D-14195, Germany. 3Institut de Chimie des Substances Naturelles, UPR 2301 CNRS, and IMAGIF CTPF and qPCR-Platform, Centre de Recherche de Gif, Gif sur Yvette, France.

SPP1 is a tailed phage that infects the Gram-positive bacterium Bacillus subtilis. Gp12 is the SPP1 capsid auxiliary protein that binds at the centre of the major capsid protein hexamers [1]. Gp12 features a repeated Gly-Xaa-Yaa (GXY) spacer between the amino and carboxyl termini regions. This motif is a signature of the collagen fold, the major animal extracellular matrix. Collagen-like repeats were identified in genes coding for bacterial and phage proteins. In phages, putative collagen-like segments where described as a part of tail proteins. We observed that gp12 is an elongated trimer in solution. The gp12 circular dichroïsm profile reveals features characteristic for the presence of collagen-like triple helices and alpha-helices. The thermal stability study of gp12 indicates that the isolated protein unfolds and dissociates reversibly with a melting temperature of 32°C, a value close to those found for eukaryotic collagen and bacterial collagen-like proteins. Gp12 binds specifically to SPP1del12 capsids, and changes dramatically the capsid surface properties. Both native gp12 trimers and unfolded gp12 monomers are able to bound to SPP1del12 capsids, but show different binding and affinity behaviours. The gp12 trimer unfolds and dissociates from SPP1 capsids at 54°C showing that association to the capsid lattice enhances its thermal stability of 22°C. Overall, this work characterizes, for the first time, a viral capsid auxiliary protein with a collagen-like fold. [1] HE. White, MB. Sherman, S.Brasilès, E. Jacquet,P. Seavers,P. Tavares, EV Orlova. J Virol. 2012

Jun;86(12):6768-77S98–100 (1918).

77

An Atomistic Model for Bacteriophage MS2 and Implications for Viral Assembly

Steve Harvey, Peter Stockley and Reidun Twarock

78

Herpesvirus Genomes, The Pressure is On

Dave Bauer1

1 Carnegie Mellon University

Herpes simplex virus 1 (HSV-1) packages its microns long double-stranded (ds) DNA genome into a nanometer-scale protein shell, termed the capsid. Due to its tight confinement within the capsid, the stiff DNA chain experiences repulsive electrostatic and hydration forces as well as bending stress. By osmotically suppressing DNA release from HSV-1 capsids, we provide the first experimental evidence of a high internal pressure of 18 atmospheres within a eukaryotic human virus, resulting from the confined genome. Capsid pressures of similar magnitude have also been found in dsDNA bacteriophages. Despite billions of years of evolution separating eukaryotic viruses and bacteriophages, pressure-driven DNA ejection has been conserved. This suggests it is a key mechanism for viral infection and thus reveals a new target for antiviral therapies.

79

Solid to fluid DNA phase-transition inside the virus. Genome metastability

Ting Liu1, Udom Sae-Ueng1, Dong Li1, and Alex Evilevitch1

1 Carnegie Mellon University, Department of Physics, Pittsburgh, PA 15213

The metastability concept most often refers to the viral capsid that needs to be sufficiently stable to protect the viral genome and unstable enough to release its genome into the cell. In this work we establish a new concept of viral metastability with regard to the viral genome. We revealed an unexpected phase-transition of the packaged dsDNA in phage λ at the temperature of infection (∼37°C), making the DNA more fluid and thus optimized for release into the cell. At the same time, at the lower temperatures, DNA behaves more like a crystal, when the conditions are less ideal for infection, thus stabilizing the viral capsid and preventing spontaneous genome release. This phase-transition can be attributed to packaged DNA going from a long-range liquid crystalline state to a more disordered state, introducing the necessary fluidity of the genome during the ejection. This observation can explain a fundamentally important question: how a single dsDNA chain compressed under 50 atm pressure can escape the capsid in seconds through a single pore with a cross section equal to that of the DNA diameter without entanglement.

80

More Than Passive Passengers: The Roles of RNA

Packaging Signals in Virus Assembly and Evolution

E.C. Dykeman1,2, P.G. Stockley3 and R. Twarock1,2

1 Department of Mathematics, University, UK

2 Department of Biology, University of York, UK

3 Astbury Centre for Structural Molecular Biology, University of Leeds, UK

Viral genomic RNA has been shown to play many critical roles during the assembly of viral capsid such as conformational switching of coat proteins (CP) [1] and the promotion of efficient assembly [2]. In bacteriophage MS2 and the plant virus STNV, the important contacts between CP and viral genomic RNA have been shown to be made by small stem-loop structures with specific sequence motifs, which we term packaging signals (PSs). PSs have now been identified in the genomes of MS2 and STNV using a combination of RNA SELEX and bioinformatics [3,4]. In light of the growing experimental evidence that the genomic RNA impacts on assembly, we have developed a new modelling paradigm which takes into account the functional roles of PSs during capsid assembly [5]. Using this model, we have shown that assembly efficiency depends on the distribution of PS affinities, suggesting that the genomic RNA sequence of a virus has an effect on overall assembly efficiency, consistent with recent experimental observations [6]. Moreover, using an in silico competitive evolution experiment between RNAs with different distributions of PSs, we have also shown that the PS affinities in the RNA evolve towards a distribution which maximizes the number of RNAs that are successfully packaged. The results of these computational models suggests that, in addition to evolving their genomes to code for proteins, RNA viruses will also be under selective pressure to ensure that their genomes contain a series of PSs with affinities that promote efficient assembly. To test this, we have developed a set of algorithms which analyse Next Generation Sequencing data from RNA SELEX (see talk by Peter Stockley) and identifies areas in the genome which are potential PSs. When combined with mutational data from multiple viral genomes, the results show that areas which encompass potential PSs are also highly conserved, suggesting that they may have a functional role and that PSs occur in a wide range of viruses including animal viruses.

[1] P.G. Stockley, et al. J, Mol. Biol. 369, 541-552 (2007). [2] K. Torapova, G. Basnak, R. Twarock, P.G. Stockley, and N. Ranson. J. Mol. Biol. 375, 824-836

(2008). [3] D.H. Bunka, et al. J. Mol. Biol. 413, 51-65 (2011). [4] E.C. Dykeman, P.G. Stockley, and R. Twarock. http://dx.doi.org/10.1016/j.jmb.2013.06.005 [5] E.C. Dykeman, P.G. Stockley, and R. Twarock. Phys. Rev. E 87, 022717 (2013). [6] A. Borodavka, R. Tuma, and P.G. Stockley. PNAS 109, 15769-15774 (2012).

81

Packaging signals in ssRNA viruses from bacteria to plants to humans

Peter G. Stockley1, Alexander Borodavka1, Amy M.Barker1, Simon J. White1, Nikesh Patel1, Neil

A. Ranson1, David J. Rowlands1, Roman Tuma1, Eric Dykeman2, Reidun Twarock2. 1 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK. 2 Departments of Biology and Mathematics & York Centre for Complex Systems Analysis, University of York, York, YO10 5DD, UK Recent single molecule (sm) fluorescence assays of in vitro reassembly of single-stranded RNA viruses from bacteria and plants have revealed the presence of multiple cognate coat protein (CP) binding sites within the viral genomes. These have a number of consequences for the efficiency and fidelity of capsid assembly. The sm assays recreate the observed in vivo RNA packaging specificity that is obscured at higher protein concentrations in in vitro reassembly. The RNA sites bound by viral CPs, termed packaging signals (PSs), have been identified using RNA SELEX. They act co-operatively to promote the correct CP-CP interactions that allow efficient formation of capsids of the correct size and symmetry. We have recently extended this analysis to other plant and human ssRNA viruses and see evidence of similar PSs in those examples. The nature of PSs and their consequences for virus assembly will be described for a number of these systems.

82

Real-time Single Molecule Fluorescence Analysis Shows that One Inactive Mutant Subunit in a Pentameric Bacteriophage

T4 DNA Packaging Motor can be Tolerated

Li Dai1, Reza Vafabaksh2, Taekjip Ha2, and Venigalla B. Rao1 1 Department of Biology, The Catholic University of America, Washington, DC 20064, USA. 2 Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, and Howard Hughes Medical Institute, Urbana, IL 61801, USA. Bacteriophage T4 packages its double-stranded DNA genome into a protein capsid using a pentameric gp17 motor assembled at the special portal vertex. Previous analysis of single packaging machines using dual optical tweezers showed that the T4 motor is one of the fastest and most powerful packaging motors reported to date. Structural and biochemical studies suggest that the motor converts biochemical energy into electrostatic force which then drives DNA translocation. Very little is known about how the ring subunits of the motor coordinate to package DNA at a rate of up to ~2000 bp/sec. We have developed a real-time single molecule fluorescence assay to quantitatively analyze the stoichiometry and coordination of actively packaging motors. DNA packaging was analyzed by immobilizing the reconstituted packaging machine complexes on a polymer covered slide. Fluorescently (Cy5) labeled short oligonucleotides and ATP were added to initiate packaging. Each actively packaging machine carrying out translocation of the fluorescent DNA was imaged in real-time using single-molecule total internal reflection fluorescence (TIRF) microscopy. To directly count the number of subunits in the packaging motor, gp17 mutants containing only a single fluorescently (Cy3) labeled cysteine residue have been constructed by removing all nine cysteines of gp17 and introducing a single cysteine at the desired position. Actively packaging machines were quantified by co-localization of both Cy3 and Cy5 labels in the same spot. Through single molecule photobleaching and colocalization analysis we observed active packaging complexes with up to five gp17 subunits but not six. Our results strongly suggest that the motor is a pentamer, in agreement with the previous Cryo-EM studies. We have constructed inactive gp17 mutants containing single Cy3 labeled cysteine and assembled them with the wild-type gp17 in different ratios. We then performed real-time fluorescence measurements by mixing the labeled inactive mutant with the wild-type gp17 and observing the packaging of Cy5 labeled DNA. By directly probing the activity of individual packaging complexes and simultaneously counting the number of inactive mutant subunits in the co-localized spots, we found that the T4 packaging motors containing a “dead” mutant subunit can still initiate packaging and translocate DNA. However, the mutant motors showed a longer lag time for packaging initiation and fewer molecules of DNA were packaged into each head, when compared to the wild-type motors. These results suggest that, unlike the ring type helicase motors and the phi29 packaging motor, the T4 motor is not strictly coordinated. DNA translocation appears to occur through localized pulses of ATP binding, hydrolysis, and translocation at the subunit level rather than at the motor level. When a defective subunit is encountered, the motor might pause transiently to recover and switch to another subunit and continue translocation.

83

Structure of a dengue virus temperature-induced fusogenic intermediate

Xinzheng Zhang,1 Ju Sheng,1 Pavel Plevka,1 Richard J. Kuhn,1 Michael S. Diamond,2 and

Michael G. Rossmann1

1 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. 2 Departments of Pathology & Immunology, Medicine (Infectious Diseases), and Molecular Microbiology,

Washington University School of Medicine, Saint Louis, MO 63110, USA We show, using cryo-electron microscopy, that although at room temperature dengue virus has a smooth surface, a diameter of ~500Å and little exposed membrane, at 37ºC the virions have a bumpy appearance with a diameter of ~550Å and some exposed membrane. The bumpy structure at 37ºC was found to be similar to the previously predicted structure of an intermediate between the smooth mature and fusogenic forms. As humans have a body temperature of 37ºC, the bumpy form of the virus would be the form present in humans. Therefore, effective vaccines need to be designed to induce production of antibodies directed against the bumpy form of the virus.

84

DNA confined inside the bacteriophage capsid. What can we

learn from cryo-EM?

A. Leforestier1, F. Livolant1

1 Laboratoire de Physique des Solides, CNRS UMR 8502, Université Paris Sud, 91405 Orsay, France

Cryo-EM is especially well adapted to the understanding of phage and virus structure. 3D reconstruction methods based on image averaging have proved very powerful for the understanding of their proteic machinery at near atomic resolution [1]. However this approach is more limited when it comes to the organisation of dsDNA in bacteriophages, which fold is unique to each individual. 3D reconstructions based on icosahedral symmetry origin a series of concentric shells at the capsid periphery, the inner volume appearing disordered. Asymetric reconstructions are a priori more informative, but still only describe the averaged local orientation/localisation of the DNA molecule. Most of them have been done on phages that possess a large inner cylindrical core [2-5]. Then, concentric ring patterns are obtained in the portal and core region. In some cases, this approach has allowed the precise localisation of terminal DNA segments [6]. In the absence of a core, the asymmetric reconstruction of phages phi29 and T4, show concentric shell patterns similar to those using icosahedral symmetry [6-8]. The loss of information coming from the averaging can be overcome by the analysis of individual 2D images, although these cannot provide a complete 3D view. We analyse 2D images of bacteriophage T5 and SPP1 containing various amounts of DNA under different physico-chemical conditions (ionic environment, temperature, osmotic pressure). DNA in the full-filled capsid is insensitive to these parameters. It is organised into monocrystalline domains separated by a lattice of defects walls, forming a structure analogous to a Twist Grain Boundary liquid crystal, with no evidence of a disordered core region. This structure is compatible with the averaged 3D reconstruction: the concentric shells correspond to the lattice planes of the liquid crystal (and allow a precise measurement of the lattice parameter); the disordered core would result from the delocalisation of the central defects. In partially filled capsids, DNA undergoes considerable conformational changes with varying the experimental conditions. Chains may occupy all the available volume (forming liquid crystalline of disordered structures), be wounded at the capsid periphery, be condensed or only partially condensed into toroids [9]. This fascinating polymorphism is still far from being wholly explored. It is likely that its characterisation would benefit of a combination of 2D and 3D analyses.

[1] Chang J., et al. (2012) Adv Exp Med Biol. 726, 49-90 [2] Jiang W. et al. (2006) Nature 439, 612-6. [3] Chang J. et al. (2006) Structure 14, 1073-82. [4] Leiman PG. et al. (2007) J. Mol. Biol. 371, 836-849. [5] Liu X. et al. (2010) Nat. Struct. Mol. Biol. 17 830-837. [6] Tang J. et al (2008) Structure 16, 935-943. [7] Comolli LP. Et al (2008) Virology, 371, 267-277. [8] Fokine et al (2004) Proc . Natl. ZAcad. Sci. USA 101, 6003-6008. [9] Leforestier A. (2013) Journal of Biological Physics, in press.

85

Structure and Dynamics of Picornavirus to Inform Vaccine

Design

A. Kotecha1, J. Seago2, K. Scott3, A. Burman2, C. Porta1, S. Loureiro4, T. Jackson2, F. Maree3, R.Esnouf1, E. Fry1, I. Jones4, B. Charleston2 and D. Stuart1

1 Division of Structure Biology, University of Oxford, United Kingdom. 2 The Pirbright Institute, Pirbright, United Kingdom. 3 Agriculture Research Centre - Onderstepoort Veterinary Institute, South Africa. 4 University of Reading, Reading, United Kingdom.

The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses which impact on human and animal health. Current picornavirus vaccines are frequently produced from inactivated virus. Inactivation often reduces the stability of the virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly above 37°C, destroying their effectiveness in eliciting a protective immune response. As a result, vaccines require a cold chain for storage and animals need to be frequently immunised. FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of antigenic variation. We sought to rationally engineer thermo-stable FMDV capsids either as infectious copy virus or recombinant empty capsids with improved thermo-stability for improved vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular modelling, free energy calculations, X-ray crystallography, electron microscopy and various biochemical/biophysical techniques were used to design and help characterise the capsids. For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were characterised by MD. Promising candidates were then engineered and shown to confer increased thermo- and pH-stability. Thus, in silico predictions translate into marked stabilisation of both infectious and recombinant empty viral capsids. A novel in-situ diffraction method was used to determine crystal structures for quality assessment and to verify that no unanticipated structural changes have occurred as a consequence of the modifications made. The in-situ technology is a quick and easy method for collecting room temperature data, avoiding the increased mosaicity often associated with cryo-crystallography. It also provides a contained environment facilitating safe data collection and avoiding the hazards and deleterious effects of crystal mounting. The structures of the wildtype and two of the stabilised mutants were solved at high resolution and the antigenic surfaces shown to be unchanged. Animal trials showed stabilised particles can generate improved neutralising antibody response compared to the traditional vaccines. Here, we have successfully used a structure based rational engineering approach to increase the stability of FMDV capsids without affecting the antigenic properties of the virus and demonstrated the direct application of structural biology and structure based design that has the potential to lead directly to a new generation of efficacious vaccines that can provide hope that the disease can be brought under control.

86

The crystal structure and the stability of C-terminal fragment of gp34, proximal half of long tail fiber of bacteriophage T4

Meritxell Granell 1, Mikiyoshi Namura 2, Sara Alvira-de Celis 3,

Carmela Garcia-Doval 3, Abhimanyu K. Singh 1, Mark J. van Raaij 3, Fumio Arisaka 2, Shuji Kanamaru 2

1 Departmento de Estructura de Macromoleculas, Centro Nacional de Biotecnologia (CNB-CSIC), calle Darwin 3, E-28049 Madrid, Spain. 2 Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-9 4259 Nagatsuta-cho, Midori-ku Yokohama 226-8501, Japan. 3 Departmento Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain.

The long tail fiber of bacteriophage T4 consists of 4 proteins, gp34, gp35, gp36 and gp37. Six long tail fibers function as a sensor for phage infection. When the tip of gp37 recognizes the surface of the host E. coli, the signal transfers to the phage baseplate through gp34. It induces the conformational change of the baseplate which triggers the phage infection. Gp34 is trimeric fibrous protein which constitutes proximal half of the log tail fiber. Here we solved the crystal structure of C-terminal 509 residues of gp34 fiber (gp34C781-1289) which is close to the hinge of long tail fiber. Overall structure is rod-shape with humps, 285 Å long and 50 Å wide (narrow shaft part 20 Å wide). The structure consists of three shaft domains and 4 globular domains. The shaft domains are all 3-stranded β- helices which have been found in the shaft of short tail fiber (gp12) of phage T4. The globular domains form “humps” with exposed loops. These loops interact with gp18, when the crystal structure was fitted into the tail fiber density in the phage tail structure which has been obtained by cryo-EM reconstruction. We also mapped the trypsin sensitive Arg and Lys residues on the crystal structure and found all the residues are located at the loops in globular domains.

87

Atomic structure of Cucumber Necrosis Virus and the Role of the Capsid in Vector Transmission

Ming Li1, Kishore Kakani2, Umesh Katpally1, Sharnice Johnson1,

D’Ann Rochon2, and Thomas J. Smith1

1 Donald Danforth Plant Science Center, 975 North Warson Road, Saint Louis, MO 63132 2 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, 4200 Hwy. 97, Box 5000, Summerland, British Columbia, Canada V0H 1Z0

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome packaged in a T=3 icosahedral particle. CNV is transmitted in nature via zoospores of the fungus, Olpidium bornovanus. CNV undergoes a conformational change upon binding to the zoospore that is required for transmission and specific polysaccharides on the zoospore surface have been implicated in binding. To better understand this transmission process, we have determined the atomic structure of CNV. As expected, being a member of the Tombusvirus genus, the core structure of CNV is highly similar to TBSV, with major differences lying on the exposed loops. Also, as was seen with TBSV, CNV appears to have a calcium binding site between the subunits around the quasi 3-fold axes. However, unlike TBSV, there appears to be a novel zinc binding site within the β-annulus formed by the N-termini of the three C subunits at the icosahedral 3-fold axes. Two of the transmission defective mutants map immediately around this zinc binding site. The other transmission and particle formation defective mutants are mapped onto the CNV structure and it is likely that a number of the mutations affect zoospore transmission by affecting conformational transitions rather than directly affecting receptor binding.

88

An atomic model of the T=9 procapsid of phage D3

Robert L. Duda1, James F. Conway,2 Matthijn Vos,3 & Roger W. Hendrix1

1 Dept. of Biological Sciences, School of Arts & Sciences, University of Pittsburgh, USA 2 Dept. of Structural Biology, School of Medicine, University of Pittsburgh, USA 3 Apps Lab, FEI Europe B.V., Eindhoven, The Netherlands

The HK97 capsid protein fold has been observed in a wide variety of capsids with different sizes and shapes from dsDNA tailed bacteriophages. Although these capsids are all constructed with common principles of icosahedral and prolate symmetry, we do not know how the different sizes and shapes are specified by variants of the common fold. Available structure of capsid proteins revealed at atomic resolution show that the main features of the fold are conserved, but extra (or missing) loops and domains abound, confounding meaningful comparisons between examples that are too structurally divergent. Our approach is to find and compare capsids that have different sizes but nearly identical folds so as to minimize distracting features. Given those criteria, we have chosen Pseudomonas phage D3 and Burkholderia phage phi1026b for our efforts in comparative capsidomics because their capsid proteins are closely similar to that of HK97 in sequence but assemble into structures with Triangulation number T=9, larger than the T=7 of the HK97 capsid. The existing library of HK97 capsid crystal structures in different assembly and maturation states provides a good set of starting models for comparison with D3. An excellent cryo-EM data set of the Prohead II form of phage D3, collected on an FEI Krios microscope equipped with a Falcon direct-detect camera and running the EPU automatic data collection software, has yielded a ~4 Ångstrom resolution map in which most of the main chain and many of the side chains can be easily discerned. We are using MDFF (molecular dynamics flexible fitting) and other routines to produce and refine a high quality model of D3 Prohead II and have begun our analyses and comparisons to the crystal structures of HK97 proheads.

89

Delivery of vaccine genes and proteins into mammalian cells using the bacteriophage T4 DNA packaging machine

P. Tao1, M. Mahalingam1, B. Marasa1, Z. Zhang1, A.K. Chopra2, and V.B. Rao 1

1Department of Biology, The Catholic University of America, Washington, DC 20064, USA 2 Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, 77555, USA The phage T4 DNA packaging machine consists of a molecular motor assembled at the portal vertex of an icosahedral head. The ATP-powered motor packages the 56µm-long 170-kb viral genome into 120nm x 86nm head to near crystalline density. We engineered this machine to deliver genes and proteins into mammalian cells. DNA molecules were translocated into emptied phage head and its outer surface was decorated with proteins fused to outer capsid proteins, Hoc and Soc. T4 nanoparticles carrying reporter genes, vaccine candidates, functional enzymes, and targeting ligands were efficiently delivered into cells, or targeted to antigen-presenting dendritic cells, and the delivered genes were abundantly expressed in vitro and in vivo. Mice delivered with a single dose of F1-V plague vaccine containing both gene and protein elicited robust antibody and cellular immune responses. This “progene delivery” approach might lead to new types of genetic therapies and prime-boost vaccines against complex infectious diseases such as HIV and malaria.

90

Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of

bacteriophage T7

F. Guo1, Z. Liu1, F. Vago1, Y. Ren1, W. Wu1, E.T. Wright2, P. Serwer2, W. Jiang1 1 Purdue University, West Lafayette, Indiana 47907, USA 2 University of Texas Health Science Center, San Antonio, Texas 78229, USA Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex [1]. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously in the average structure of all particles. Our results imply that layers with mis-matched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. By progressively further classifying particles into subpopulations, distinct structures of the various isomers are separately reconstructed. In these isomer structures, not only the two combinations of three neighboring distinct symmetries (5-12-8 and 12-8-4) but also all four distinct symmetries (5-12-8-4) can be simultaneously resolved. When successively viewed, the structures of these isomers collectively generate apparent rotation of the core stack around the portal axis. The properly resolved structures of the isomers allowed us to find a small tilt of the core stack relative to the icosahedral five-fold axis. We propose that the tilted core stack can undergo a precession, similarly to that of the ribbon exercise in rhythmic gymnastics, to assist DNA spooling without tangling during packaging. [1] F. Guo, Z. Liu, F. Vago, Y. Ren, W. Wu, E.T. Wright, P. Serwer, W. Jiang. Proceedings of the National Academy of Sciences 110, 6811 (2013)

91

DNA Length Quantization in Phage T3/T7 DNA Packaging and Expulsion

Philip Serwer1, Elena T. Wright1, Fei Guo2 and Wen Jiang2

1 The University of Texas Health Science Center, San Antonio, Texas, USA 2 Purdue University, West Lafayette, Indiana, USA Past studies revealed that DNA packaging of phages phi29, T3 and T7 sometimes produced incompletely packaged DNA (ipDNA) that formed mysteriously sharp bands during gel electrophoresis (ipDNA quantization). Past studies of packaging kinetics revealed that phage lambda also underwent mysteriously quantized DNA packaging [review of three independent studies is in Serwer, P. and Jiang, W. Bacteriophage (2012) 2, 239]. We discover a packaging ATPase-free, in vitro model for understanding T3 ipDNA quantization. First, we use selection for propagation in high [NaCl]-media to isolate a multi-site T3 mutant that hyper-produces tail-free capsids with mature DNA (heads). Mutations are in early, DNA replication and tail protein genes; none are in head protein genes. A variable-length DNA segment leaks from some mutant (but not wild type) heads, based on DNase I-protection assay, confirmed by electron microscopy. The length of the protected DNA segment is quantized, based on restriction endonuclease analysis, which produces six sharp bands of DNA missing 3.7-12.3% of the last end packaged (left end) of the 38.2 Kb genome. The separation between band-forming DNAs is 0.2-1.2 Kb (~0.41 Kb packaging steps for lambda). Native gel electrophoresis confirms quantization of DNA expulsion and reveals that some protein shells of DNase I-digested heads have undergone contraction. The contraction extent suggests that shell radius is the ruler for ipDNA quantization. Linkage of quantized DNA expulsion to packaging-associated ipDNA quantization is suggested by increase in mutant ipDNA production (no DNase I used) as we increase production of mutant heads by decreasing [NaCl] in the growth medium. A possible evolutionary basis for DNA quantization is programmed stalling that provides time for feedback control during DNA packaging and injection. We note that our T3 heads with partially expelled DNA are the first stable (for > 6 months) particles of this type, to our knowledge. Physically, these particles are analogs to DNA packaging intermediates. The above study also shows that native gel electrophoresis can be used to detect hard-to-isolate DNA packaging intermediates with some DNA packaged and some outside of the capsid. Finally, cryo-EM reveals yet another shell state when used for 3D reconstruction of a T7 DNA packaging intermediate called MLD (Metrizamide low density) capsid II. This capsid is produced during in vivo DNA packaging and is isolated via its low density during buoyant density centrifugation in a Metrizamide density gradient. The low density is caused by the impermeability of the gp10 shell and gp8 connector to Metrizamide. We find that the gp10 shell of T7 MLD capsid II is 1.8% larger than the gp10 shell of T7 phage (related to T3). This difference is caused by 1.2° rigid-body rotation of gp10 asymmetric units in each triangular face of the icosahedral shell. The subunits are HK97-like in conformation. The T7 MLD capsid II reconstruction is the first of a hyper-expanded shell of a double-stranded DNA phage. One of us (PS) has predicted that even more hyper-expansion occurs for gp10 shells when a back-up cycle of the DNA packaging motor activates. This work was supported by NIH (R01AI072035) and the Welch Foundation (AQ-764).

92

A Mechanistic View of Bacteriophage Headful Packaging

Paulo Tavares1, Rudi Lurz2 and Tom Trautner2

1Unité de Virologie Moléculaire et Structurale, CNRS UPR 3296 and IFR 115, Bât. 14B, CNRS, 91198 Gif-sur-Yvette, France 2 Max-Planck Institut für Molekulare Genetik, Ihnestrasse 73, D-14165 Berlin, Germany

In tailed bacteriophages and in herpes viruses, a viral ATPase/endonuclease (terminase) bound to DNA docks in the specialized portal vertex of the procapsid to assemble the packaging motor. The motor translocates DNA through the portal protein tunnel to reach high concentration inside the viral capsid. In numerous phages the substrate DNA molecule is cut when a threshold amount of DNA is reached inside the capsid (headful cleavage). Mutations affecting the headful sensor were mapped exclusively in the portal protein demonstrating its central role in the mechanism that triggers the switch of terminase activity from translocase to endonuclease. We report genetic and biochemical studies from the past 20 years whose mechanistic implications are interpreted in light of recent structures of the SPP1 portal system. Mutations in different positions of the portal polypeptide chain have an additive undersizing phenotype on the length of packaged DNA. Each individual portal subunit contributes to the sensor trigger that leads to headful cleavage. This information together with positioning of mutations in the SPP1 portal protein structure that affect DNA packaging efficiency and/or the sensor detection threshold provide a framework to interpret the headful mechanism molecular basis.

93

A Mechanistic View of Bacteriophage Headful Packaging

Michael Feiss1

1 University of Iowa

Lambda-like phage N15’s packaging system differs from that of lambda in (1) having simpler cosB recognition and (2) cohesive end bp changes at positions 9 and 12. The lambda versus N15 cohesive end mismatch reduces virus fitness about two-fold. Virus chromosomes with the cohesive end mismatch cyclize with reasonable efficiency, and the mismatch can persists until chromosome duplication. Virus yield studies suggest that the endonuclease activity of terminase acts with equal efficiency at the cosNs of lambda and N15. Scanning mutagenesis across N15 bps 42-to-199 identifies a single segment, bp 48-59, that is essential for efficient N15 DNA packaging. The packaging specificities of lambda, 21, and N15 have been compared. Like lambda, N15-specific terminase does not recognize phage 21 DNA for packaging. Although lambda-specific terminase does not package N15 DNA, N15-specific terminase surprisingly sponsors packaging of N15 DNA. Evolution of packaging specificity in the lambda-like phages will be discussed

94

Shedding light on the early stages of RNA packaging by

single molecule fluorescence

R. Tuma1, A. Borodavka1, P.G. Stockley1

1 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK

Genome encapsidation is an essential step in the morphogenesis of RNA viruses. During this process viral cognate RNA is selected from the pool of potential cellular competitors and condensed to fit into the confines of the virion. In order to monitor the conformational changes during packaging we have labeled various genomic as well as cellular competitor RNAs with fluorescent dyes. Fluorescence correlation spectroscopy was used to estimate hydrodynamic sizes of RNA molecules at low concentrations, i.e. close to infinite dilution, in order to minimize non-specific interactions and aggregation [1]. While genomic RNAs of ssRNA viruses are generally compact they are still substantially larger to fit into the inner volume of their cognate capsids. Interestingly, some cellular mRNA and ribosomal RNA are also similarly compact, raising question about how viruses discriminate between viral and cellular RNA. Using in vitro assembly assays for two ssRNA viruses (phage MS2 and plant virus STNV) we have detected a rapid, cooperative collapse in viral RNA size upon addition of cognate coat protein [2]. Cellular or non-cognate viral RNA did not undergo such collapse, suggesting packaging specificity is exercised at this stage. We have also investigated early stages in packaging of segmented dsRNA viruses of the Reoviruidae family (human rotavirus, avian reovirus). Packaging in these viruses starts with ssRNA genomic precursors being incorporated into viral cores and replicated. The process is aided by interaction between viral non-structural proteins (reovirus sigmaNS or rotavirus NSP2) and the RNA precursors. Prior to such interaction rotavirus segments are less compact than the genomes of ssRNA viruses exhibiting similar length, presumably due to their different mode of packaging.. Upon addition of sigmaNS the resulting complex is less compact than the corresponding RNA with dramatically reduces amount of secondary structure. This suggests that the role of non-structural proteins is in early stages of segment assortment rather than in condensation into the virion.

[1] A. Borodavka, R. Tuma, and P. G. Stockley RNA Biol. 10, 481–489 (2013). [2] A. Borodavka, R. Tuma, and P. G. Stockley Proc Natl. Acad. Sci. USA 109, 15769-15774 (2012).

95

Poster Abstracts

96

A.1 Characterization of archaeal spindle-shaped virus His1 and its comparison to other short-tailed spindle-shaped

viruses

Nina Atanasova1, Maija Pietilä1, Hanna Oksanen1, and Dennis Bamford1

1 Institute of Biotechnology, University of Helsinki, PO Box 56, Viikinkaari 5, Helsinki 00014, Finland Viruses of the archaea have been studied for the last four decades. In spite of that only approximately 100 archaeal viruses have been described in more detail. Structurally these viruses represent either morphotypes that are known for bacterial and eukaryotic viruses (head-tailed and tailless membrane containing icosahedral viruses), or unique structures that are specific only for archaeal viruses (e.g. pleomorphic, bottle-shaped and spindle-shaped virions). Extreme environments, such as hyperthermophilic or hypersaline waters are rich in archaeal viruses and especially high counts of spindle-shaped virus-like particles have been observed. To date, less than 20 spindle-shaped archaeal viruses or virus-like particles (VLPs) are known, which have been classified into two viral families Fuselloviridae (virions with one short tail) and Bicaudaviridae (virions with one or two long tails) and one genus, Salterprovirus (virions with one short tail). Most of these viruses infect hyperthermophilic archaea, and only one virus, His1, has been so far described for extreme halophiles. We performed life cycle studies and biochemical characterization of His1 revealing that the virus is non-lytic and can tolerate a range of different salinities (0.1-4.1 M NaCl). The virus was shown to have only one major structural protein, which is likely lipid-modified, but His1 virion does not seem to contain a lipid membrane bilayer. Due to both genomic and structural similarities, His1 and fuselloviruses might be related and possibly have a common origin.

97

A.2 Cryo-EM of Portal Machinery of Bacteriophage PRD1

Dennis Bamford1 1 Institute of Biotechnology, University of Helsinki, PO Box 56, Viikinkaari 5, Helsinki 00014, Finland PRD1 is an icosahedral dsDNA bacterial virus with an inner membrane (Tectiviridae family). Based on previous X-ray crystallographic results of PRD1, its major capsid protein has similar fold as those of several other viruses such as adenovirus, PBCV-1 and STIV. However, the structure of the non-icosahedrally arranged portal complex anchored in the inner membrane remains elusive. Biochemical and immuno-electron microscopic studies have identified four proteins in the portal complex: the packaging ATPase P9, the packaging efficiency factor P6, and the integral membrane proteins P20 and P22. The goal of our study is to reveal the structures of the portal machinery of PRD1. We used single-particle electron cryo-microscopy (cryo-EM) to study the mature virion and three procapsid mutants of PRD1 using symmetry-free reconstructions. Their density maps allow us to conclude the locations and features of the four portal proteins at a unique vertex. The P20 and P22 form a hexamer of dimers embedded in the viral membrane and function as a conduit for the DNA packaging. The P20 or P22 cannot exist alone without the other. The P6 and P9 form a 12-mer of a portal complex with ATPase activity similar to other phage portal protein complex. This is the first structural evidence of the PRD1 packaging complex operating at a specific vertex, and shows the connection between the membrane and the capsid shell providing a conduit for DNA translocation in an ATPase-driven reaction.

98

A.3 The interaction of phage HK620 with its LPS receptor on

host cell surface

N. K. Broeker1, D. Andres2, R. Seckler1, S. Barbirz1

1 Physical Biochemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14471 Potsdam, Germany 2 Present Adress: Harvard University, Department of Chemistry and Chemical Biology, Kahne Laboratory, 12 Oxford Street, Cambridge, MA 02138, USA

In Gram-negative bacteria the rigid outer membrane displays a protective barrier. However, bacteriophages overcome this barrier and use lipopolysaccharides (LPS) for initial attachment. In our group we work on different bacteriophages recognizing these LPS structures in the first step of infection. For this recognition of the LPS the tailspike proteins (TSP) of these phages are essential. With podovirus P22 and siphovirus 9NA two phages of different morphologies infecting the same Salmonella enterica hosts were already described by our group regarding the infection mechanism. Andres et al. could show that in both phages the host LPS is the only receptor needed to trigger the DNA ejection in vitro and that TSP activity is required [1,2]. To prove if the provided mechanism for P22 could be common for other podoviruses in vitro DNA ejection studies with the homologous E. coli phage HK620 were carried out. The specificity of this phage was studied as well as features necessary for DNA ejection. In vitro this related phage HK620 behaves differently compared to P22. We found that for successful DNA ejection in these in vitro studies the LPS morphology in solution is crucial. Thus, differences concerning the properties of the LPS from P22 and HK620 host cells lead to diverse results in in vitro DNA ejection experiments. However, we could show that the host cell LPS is the only receptor needed to trigger DNA ejection in HK620 as demonstrated before for P22.

[1] D. Andres, C. Hanke, U. Baxa, A. Seul, S. Barbirz, R. Seckler. J Biol Chem. 285(47) (2010). [2] D. Andres, Y. Roske, C. Doering, U. Heinemann, R. Seckler, S. Barbirz. Mol Microbiol. 83(6) (2012).

99

A.4 Maturation of T5 capsid: a view throughout in vitro

expansion and decoration

O. Preux1, D. Durand1, A. Huet1,2, J. F. Conway2, M. Renouard1, A. Bertin1, P. England4, F.

Wien3, J. Pérez3, P. Vachette1, P. Boulanger1

1 Institut de Biochimie Biophysique Moléculaire et Cellulaire, UMR 8619, Univ. Paris Sud, Orsay, France

2 Department of Structural Biology, University of Pittsburgh School of Medicine, USA 3 Synchrotron SOLEIL, F-91192 Gif sur Yvette, France 4 Plate-Forme de Biophysique des Macromolécules et de leurs Interactions, Institut Pasteur, Paris, France

Capsids of dsDNA bacteriophages are extraordinarily robust macromolecular assemblies capable of withstanding the strong internal pressure generated by the tightly packed DNA they contain. They initially assemble into compact procapsids, which undergo expansion upon genome packaging. This shell remodeling results from a structural rearrangement of head protein subunits that yields highly stable mature capsids. The expansion process of the large capsid of bacteriophage T5 (T=13) was investigated in vitro by using the intermediate prohead II form, which is competent for packaging the 121 kbp dsDNA genome. Prohead II morphology and dimensions were characterized by cryo-electron microscopy and small angle X-ray scattering. Decreasing the pH or the ionic strength triggers expansion of prohead II, converting them into thinner and more faceted capsids isomorphous to the mature virion particles. Prohead II expansion is a highly cooperative process lacking any detectable intermediate. This two-state reorganization of the capsid lattice leads to a remarkable stabilization of the particle, with no need for reinforcement by inter-subunit crosslinking or additional cementing proteins. The expanded T5 capsid can be decorated by the purified protein pb10 (monomers of 17.3 kDa), which binds with a very high affinity and in a cooperative way to the centre of hexamers of the pb8 protein. These features make T5 a very attractive model for investigating the mechanism of the allosteric transitions that take place upon maturation of dsDNA bacteriophage capsids.

100

A.5 Comparing the common structural subunits of phage phi1026b and HK97 by high resolution cryo-electron

microscopy

Patricia Campbell1

1 University of Pittsburgh Phage HK97 is a lamba-like virus of E. coli that has been extensively characterised. The entire icosahedral capsid structure has been solved crystallographically and the fold of its major capsid protein is now suspected to be shared by the extremely large family of dsDNA tailed phages, and by herpesviruses. However, it is unknown how this fold is exploited to form the large variety of capsid sizes and shapes observed in this family. Details of specific capsid architectures and functions are essential for targeting viral capsid components by novel antiviral therapies, such as for herpesviruses, and for understanding the evolutionary connections between structurally related capsids. One of our focuses is phage phi1026b whose major capsid protein is 50% identical to that of HK97 but whose assembly pathway yields a larger capsids composed of 540 copes arranged with an icosahedral triangulation number, T=9, compared to the 420 copies of the T=7 HK97 capsid. Recent developments in cryo-electron microscopy have allowed us to generate near-atomic resolution models of procapsids that are early in the assembly pathway, and in these are resolved a-helices with detectable chirality, separation of B-strands, and possible identification of large sidechains for well-placed amino acids. The crystal structure for the HK97 capsid protein has been adjusted to the phi1026b cryoEM map by substituting the endogenous residues and flexibly-fitting the fold to the cryoEM density using a molecular dynamics approach called MDFF. The cryoEM density well matches the major features of the HK97 fold and provides powerful constraints for adjusting loops to match the phi1026b structure. Further modelling of phi1026b density that is additional to the HK97 model was completed with COOT. Modelling and interpretation are underway and we expect to achieve a highly plausible pseudo-atomic model of the phi1026b capsid that may be compared to the HK97 crystal structure to address the question of what in the capsid protein fold governs capsid geometry.

101

A.6 Visualizing virus assembly intermediates inside marine cyanobacteria

W. Dai1, C. Fu1, D.A. Raytcheva2,3, J. Flanagan1, †, H.A. Khant1, X. Liu1,

R.H. Rochat1,4, C. Haase-Pettingell2, J.M. Piret3, S.J. Ludtke1,4, K. Nagayama5, M.F. Schmid1,4, J.A. King2 and W. Chiu1,4

1 National Center for Macromolecular Imaging, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030 USA. 2 Massachusetts Institute of Technology, Cambridge, MA 02139 USA. 3 Department of Biology, Northeastern University, Boston, MA 02115 USA. 4 Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, 77030 USA. 5 Division of Nano-structure Physiology, Okazaki Institute for Integrative Bioscience, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan † Present address: FEI, 5350 Dawson Creek Drive, Hillsboro, OR 97124, USA

Cyanobacteria are photosynthetic organisms responsible for ~25% of organic carbon fixation on earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen billions of years ago. Cyanophages, which infect these bacteria, play an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer [1, 2]. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike Phase Contrast (ZPC) electron cryo-tomography (cryoET) [3, 4]. This imaging modality yields significant enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identified distinct Syn5 assembly intermediates. Our results suggest that the phage releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in assembly, we detected full particles with a tail and then with an additional horn. The morphogenetic pathway we describe herein is highly conserved and was probably established long before that of double stranded DNA (dsDNA) viruses infecting higher life forms.

[1] W.H. Pope, et al. J. Mol. Biol. 368, 966-981, (2007). [2] D.A. Raytcheva, C. Haase-Pettingell, J.M. Piret, & J.A. King, J. Virol. 85, 2406-2415, (2011). [3] R. Danev & K. Nagayama, Methods Enzymol. 481, 343-369, (2010). [4] K. Murata, et al. Structure 18, 903-912, (2010).

102

A.7 Mechanisms of Termination of Bacteriophage DNA

Packaging Studied with a Single-molecule Optical Tweezers

Measurement

D. delToro1, J. Sippy2, M. Feiss2, and D.E. Smith1

1 University of California San Diego, 9500 Gilman Drive, La Jolla, CA, USA 92093 2 University of Iowa, 5 W Jefferson St, Iowa City, IA, USA 52245

The genomes of many dsDNA viruses are replicated by a mechanism that produces a long concatemer of multiple genomes. These viruses utilize a multifunctional molecular motor complex referred to as "terminase" that can initiate packaging at the appropriate start point, translocate DNA, sense when a single genome has been packaged, and then switch to a mode that arrests packaging and cleaves the remaining DNA concatemer to release a filled virus head [1,2]. We have recently developed an improved method to measure single phage Lambda DNA packaging using dual-trap optical tweezers and prestalled motor-DNA-procapsid complexes. We are applying this method to test proposed mechanisms for the sensor that triggers termination; specifically a velocity-monitor vs. energy-monitor vs. capsid-filling monitor. Preliminary results using DNA templates with an ectopic termination site and mutant terminase exhibiting slowed DNA translocation velocity suggest termination is not triggered by a velocity-monitor. [1] C.E. Catalano. Viral Genome Packaging Machines: Genetics, Structure and Mechanism, New York: Springer US (2005) [2] Rao, V. B., Feiss, M. Annual Review of Genetics, 647-681 (2008).

103

A.8 The molecular architecture of the bacteriophage T4 neck

A. Fokine1, Z. Zhang2, S. Kanamaru1,3, V.D. Bowman1, A.A. Aksyuk1, F. Arisaka3, V.B. Rao2,

M.G. Rossmann1

1 Purdue University, Department of Biological Sciences, West Lafayette, Indiana, 47907, USA 2 The Catholic University of America, Department of Biology, Washington, DC, 20064, USA 3 Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology, Yokohama, 226-8501, Japan

The bacteriophage T4 tail terminator protein, gp15, attaches to the top of the phage tail stabilizing the contractile sheath and forming the interface for binding of the independently assembled capsid. We present the X-ray structure of the gp15 hexamer, describe its interactions in T4 virions, and discuss its structural relationship to other phage proteins [1]. We show that electrostatic forces play an important role in the T4 neck assembly.

The neck of T4 virions is decorated by the "collar" and "whiskers", made of fibritin molecules. Fibritin helps to attach the long tail fibers to the virus during the assembly process. The collar and whiskers are environment-sensing devices, regulating the retraction of the long tail fibers under unfavorable conditions, thus preventing infection. Cryo-electron microscopy analysis suggests that twelve fibritin molecules attach to the phage neck with six molecules forming the collar and six molecules forming the whiskers [1].

[1] A. Fokine, Z. Zhang, S. Kanamaru, V.D. Bowman, A.A. Aksyuk, F. Arisaka, V.B. Rao, M.G. Rossmann. J Mol Biol. 425, 1731 (2013)

104

A.9 Bacteriophages in engineered bioreactors and natural systems-phage bacteria interactions

Ananda Shankar Bhattacharjee1 and Ramesh Goel1

1 Department of Civil and Environmental Engineering, University of Utah, Salt Lake City, UT 84112

Bacteriophages are abundant and exist wherever bacteria reside. Engineered bioreactors such as municipal wastewater treating bioprocesses and natural systems such as wetlands and lakes are not exceptions because these systems are either bacteria dependent or bacteria dominated. Although, phage-host interactions in natural systems (i.e. marine environment) have been well studied, the efforts have not included natural systems like wetlands and streams. Furthermore, specific details on lytic and lysogenic cycles in engineered bioreactors and how these cycles play a role in the overall process efficiency are missing. Our lab is engaged in researching phage diversity in engineered systems. We are also investigating host-phage interactions in wetlands and streams and, using phage genomics to reveal phage diversity. This poster presentation will include some of the key findings from this research in our lab.

105

A.10 Capsid-locking mechanism in the maturation of a T=4

virus

B.C. Goh1, B.M. Kearney2, J.E. Johnson2 and K. Schulten1

1 University of Illinois at Urbana-Champaign, 405 N. Mathews Ave, Urbana, IL 61801 USA 2 The Scripps Research Institute, 10550 N. Torrey Pines Road MB-31. La Jolla, CA 92037 USA

A virus must mature before it can infect its host cell. To investigate this maturation process, an insect-infecting icosahedral virus, the Nudaurelia capensis ω Virus (NωV) was selected as a model system. Previous studies have demonstrated that the virus capsid of NωV undergoes a large conformational change (LCC) and autoproteolysis when the environmental pH changes from 7.6 to 5.0 [1]. As a result, the capsid shrinks from 480Å to 410Å in diameter.

Prior studies have also shown that it is autoproteolysis, rather than the LCC, that is the essential step to complete the maturation of NωV [1,2], and it is thought that the gamma peptides resulting from autoproteolytic cleavage are important for “locking” the shrunken capsid [2].

Using homology modeling and molecular dynamics, we constructed all-atom, shrunken capsid structures and simulated them to investigate the capsid-locking mechanism of cleaved gamma peptide. By applying a radial expansive force on the capsid of a NωV in which the gamma peptide had been deleted, we showed that the capsid exhibited a larger degree of expansion compared to the wild type NωV capsid. Our results provide further evidence for the capsid-locking role played by gamma peptide.

[1] M.A. Candy, H. Tsuruta, and J.E. Johnson. J. Mol. Biol. 311, 803-814 (2001). [2] J. Tang, K.K. Lee, B. Bothner, T.S. Baker, M. Yeager, and J.E. Johnson. J. Mol. Biol. 392, 803-812

(2009).

106

A.11 Virus-like particles assembled for use as respiratory

virus vaccines

Paul Gottlieb1, Diana Dalfo2, Helene Boigard2, Al Katz3, Alexandra Alimova1, Hui Wei1, and Jose

Galarza2

1 Sophie Davis School of Biomedical Education, City College of New York, New York, NY 10031 USA 2 TechnoVax, Inc, 765 Old Saw Mill River Road, Tarrytown, NY 10591 USA 3 Physics Department, City College of New York, New York, NY 10031 USA We utilize transfection of mammalian cells for the assembly and isolation of virus like-particles (VLPs) of both Influenza and respiratory syncytial virus (RSV). VLPs are promising candidates for use as human vaccines because they are highly immunogenic, unable to replicate or cause infection. Therefore inactivation is not required and the immunological properties of the antigens are better maintained. This latter quality is of particular importance with RSV as formalin treated vaccines have been shown to enhance disease rather than afford protection. Additionally, the nature of the VLP scaffold allows us not only to rapidly alter specific epitope components but also to reengineer surface antigens to display conserved subdomain sites able to elicit broadly neutralizing immune responses, an essential attribute needed to overcome antigenic variation and control emerging infectious agents in relative real-time. VLPs for high pathogenicity influenza viruses can be produced safely and rapidly in response to the emergence of a new pandemic strains, probably the best understood human disease model on a season-to-season basis. VLP-based vaccines are purified directly from cell culture medium in continuous culture systems and scalable for large volume manufacturing. Electron microscopy (EM) analysis focuses on particle morphology with identification and classification of surface proteins, hemagglutinin (HA) and neuraminidase (NA) in influenza and fusion protein F and glycoprotein G in RSV. We have demonstrated that pleomorphic VLPs are assembled in transfected cells and that they carry the viral surface spikes attached the VLP envelope. Furthermore, the assembled VLP appear to be morphological mimics of bona fide natural agents and displayed surface spikes exhibit broadly neutralizing epitopes as shown by immunogold labelling EM with specific monoclonal antibodies. The ability to safely, economically, and rapidly produce candidate human vaccine compositions with broadly neutralizing capacity, especially to viral agents in continuous genetic drift, woud have a significant inpact on healthcare in general, and a major paradigm shift in world wide control of influenza and RSV infections.

107

A.12 Investigating the DNA binding activity of the DBD of the

small terminase protein (GP1) from SPP1

Sandra Greive1, Maria Chechik1, Grigory Gladyshev1,2, Matthieu Glosseau1,3, and Fred Antson1. 1 York Structural Biology Laboratory, Department of Chemistry, University of York, York, United Kingdom 2 School of Bioengineering and Bioinformatics, Moscow State University, Moscow 119991, Russian Federation 3 M2 Architecture et fonction du vivant, Université de Strasbourg, Strasbourg, France

The small terminase protein from SPP1 initiates the packaging of the phage genome through binding to the pac sequence [1]. This protein forms a circular homomeric assembly of 9 subunits through the interaction of the c-terminal oligomerisation domains and binds DNA via the N-terminal DNA binding domain (DBD) that projects out from the core of the oligomer [2]. Although structures of the full-length protein and the DBD alone have been solved [2,3], detailed structural and functional characterisation of the protein-DNA complex remains challenging. We have characterised the interaction between the DBD and DNA using surface plasmon resonance and analytical ultracentrifugation with results suggesting a non-specific mode of binding, with a KD of ~10-5 M and a site size of 4-5 bp. Further confirmation by fluorescent anisotropy binding assays are ongoing as are crystallisation trials of the complex based on the binding mode determined above. [1] S. Chai, R. Lurz, and J. Alonso. J. Mol. Biol. 252(4): 386-98 (1995) [2] C. Buttner, M. Chechik, M. Ortiz-Lombardia, C. Smits, I-O, Ebong, V. Chechik, G. Jeschke, E. Dykeman, S. Benini, C.V. Robinson, J.C. Alonso, and A. Antson. PNAS 109 (3) : 811-6. (2012) [3] S. Benini, M. Chechik, M. Ortiz-Lombardía, S. Polier, A. Leech, M.B. Shevtsov, and J.C. Alonso. Acta. Cryst. F. 69(Pt 4) : 376-81. (2013)

108

A.13 On the mechanism of phage HK97 capsid protein

crosslinking and a novel bacteriophage

Dierkes LE1, Peebles CL1, Tso DJ1, Hendrix RW1, Duda RL1.Pope, W1.

1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA

The HK97 crosslink is an isopeptide (amide) bond between Lys 169 of one subunit and Asn 356 of the adjacent subunit. It forms autocatalytically in response to conformational changes in the subunits during capsid maturation. Glu 363 of a third subunit appears positioned to have a catalytic role in crosslink formation; we have confirmed this role using mutational changes, which also suggest that an important property of E363 in this context is its ability to accept a proton, i.e. to act as a general base [1]. Our model is that E363 accepts a proton from K169, making it a good nucleophile for attacking N356. For the proton transfer to work requires, we suggest, a non-polar environment , the last component of which is entry of Val 163 into the vicinity of the reaction site. We have used a genetic approach to establish which amino acids can (4) and cannot (15) substitute for V163. This allows us to draw inferences about how the reaction works. And now for something completely different: In the course of isolating new tailed phages that grow on cyanobacteria, we discovered a novel bacteriophage that has a virion morphology that is unlike that of any previously known bacteriophage. Characterization of this phage, which is named Waldemar, is still in the very early stages, but we note that the virion morphology is quite similar to that of the Rudiviridae. Rudiviruses are dsDNA viruses that infect certain members of the Archaea.

[1] Dierkes LE, Peebles CL, Firek BA, Hendrix RW, Duda RL. J Virol. (2009) 83(5): 2088-98.

109

A.14 Determining the end-to-end distance of long RNA

molecules

C. Beren1, M. Comas Garcia1, R. F. Garmann1, C. Siv1, C.M. Knobler1, and W.M. Gelbart1

1 University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095. USA

Long single-stranded (ss) RNA molecules (with lengths greater than 1000 nucleotides) play major roles in a wide variety of biological contexts, notably as messenger RNAs and as viral genomes. Recent theoretical modeling has elucidated generic behaviors of these molecules, especially the large scale/coarse-grained consequences of secondary structures on their statistical properties. In particular, Yoffe et al. [1] have argued – because of the large extent of base-pairing associated with almost any long sequence of nucleotides – that the ends of any long ssRNA are necessarily close (within several nanometers) of one another, independent of length or sequence. Here we test this theoretical prediction by measuring the end-to-end distances of a series of long ssRNAs, ranging in length from 500 to 3200 nucleotides (nt) and including both coding and non-coding sequences. We use splint ligation techniques to label both the 5' and 3' ends of these molecules with small gold nanoparticles [2] (AuNPs) and subsequently visualize them using cryo-electron microscopy [3]. Cryo-EM of RNA molecules in 100nm-thick vitrified thin films suspended over micron-sized holes allows us to simultaneously see both the RNA and the AuNPs in a single complex. In this way, real-space images of the entire RNA-AuNP conjugates can be constructed for a range of solution conditions, including denaturing conditions for which base-pairing interactions are weak enough so that the RNA behaves like a linear – instead of branched – polymer. We find that the end-to-end distance of long RNAs under native conditions is indeed small and independent of length and sequence, whereas the distance between ends of denatured molecules increases with the ½ power of the number of nucleotides, independent of sequence.

[1] Yoffe, A.M., Prinsen, P., Gelbart, W.M., and Ben-Shaul, A. (2011) The ends of a large RNA molecule are necessarily close. Nucleic Acids Res. 39(1), 292-99. [2] Ackerson, J.C., Sykes, M.T., and Kornberg, R.D. (2005) Defined DNA/nanoparticle conjugates. PNAS 102(38), 13383–85. [3] Gopal, A, Zhou, Z.H., Knobler, C.M., and Gelbart, W.M. (2012) Visualizing large RNA molecules in solution. RNA 18, 284-99

110

A.15 Simple Statistical Mechanical Theory for the Yield of RNA Packaging by CCMV Capsid Protein

S.W. Singaram1,2, R. F. Garmann1, C. M. Knobler1, W. M. Gelbart1 and A. Ben-Shaul2

1 Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90024, USA

2 The Fritz-Haber Research Center, Hebrew University of Jerusalem, Jerusalem 91904, Israel

A recent experimental study study [1] of the in vitro self-assembly of nucleocapsids from CCMV capsid protein (CP) and 3200nt-RNA has quantified the fraction, f, of packaged RNA as a function of CP:RNA molar ratio. In the absence of RNA, the CP exist as dimers (D) in solution. Each nucleocapsid consists of a single RNA molecule inside a shell of 90 dimers (180 proteins). For the wild-type CP dimer whose two N-termini contain 20 basic residues, the D:RNA ratio, θ, needs to be as high as 160, in order for all of the RNAs to be packaged. These 160 dimers involve a total of 3200 N-terminal cationic residues, thereby matching the total phosphate (anionic) charge of the RNA. We model the RNA molecule as a 1D lattice of 160 binding sites, each corresponding to 20 phosphates, and calculate the fraction of RNAs packaged as a function of D:RNA ratio by imposing two necessary and sufficient conditions for packaging: at least 90 dimers must be bound to the lattice and the size of a nearest-neighbor string of bound protein dimers must be at least m* (the critical nucleus size). The theoretical f(θ) is formulated as a convolution of the probability of there being at least N (≥ 90) CP dimers on an RNA (for a given value of θ) and the probability of finding a nearest-neighbor string of length at least m* (for a given value of N). The measured f is accounted for by a m* value of 5 and a nearest-neighbor dimer attraction energy, ε, of -5 kBT –for both wild-type CP dimers and for mutants with 8 and 12 N-terminal basic residues.

[1] R.D. Cadena-Neva, et. al.. J. Virology 86, 3318 (2012).

111

A.16 Development of an agrobacterial system for growing CCMV virus in cowpea plants, for studying RNA packaging in planta, and for mutating capsid protein for functionalization

of CCMV virus-like particles

Devin Brandt1, Adam Biddlecome1, Charles M. Knobler1, and William M. Gelbart1

1 University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095. USA Cowpea Chlorotic Mottle Virus (CCMV) is an icosahedral, positive-sense, RNA virus with a tri-partite genome. Each virion contains 180 capsid proteins (CP) and about 3000 nucleotides of RNA. The capsid protein has a long, flexible, positively charged, N-terminal tail that underlies its ability to package a variety of RNAs in vitro. The viral proteins, encoded in RNA genes, can be genetically modified through their corresponding CDNAs. Agrobacteria Tumefaciens is a gram-negative bacterium infecting dicotyledonous plants from over 140 distinct species. Agrobacterium is unique in its method of infection; unlike other pathogenic plant microbes, which cause damage extracellularly, agrobacteria are capable of inserting a portion of their genome into the nuclear DNA of their host. They do this in the form of a T-plasmid, subsequently hijacking the host’s own transcription/translation machinery to manufacture a suite of molecules that are critical to the bacterium’s survival.

Here we present the use of agrobacteria as a vector for the directed mutagenesis of the RNA genome of CCMV. cDNAs corresponding to the tripartite genome of CCMV have been integrated into a modified agrobacterial T-vector, which was generously provided to us by A.L.N Rao, incorporated into agrobacteria, and used to generate mutated virus in planta [1]. In particular, we have mutated an alanine on the external portion of the CCMV CP into a much more chemically active cysteine. This cysteine will be used to conjugate various targeting motifs – including antibodies to cancer cell antigens – to the outside of CCMV capsids, allowing us to further functionalize the plant viral capsid as a vector for gene and drug delivery in mammalian cells.

[1] Chaturvedi S, Jung B, Gupta S, Anvari B, Rao AL. J Vis Exp. 2012 Mar 1.

112

A.17 Analysis of nodaviral genome packaging during mixed

infection of BHK cells

Radhika Gopal1 and Anette Schneemann1 1 Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA Genome packaging and assembly of multipartite viruses is a complex process involving interactions between host and viral factors. The molecular determinants of recognition and encapsidation of multipartite genomes by viral capsid proteins are poorly understood. In this study, we used two nodaviruses Flock House virus (FHV) and Nodamura Virus (NoV), which co-package two positive-sense RNAs, RNA1 and RNA2, to study the mechanisms underlying genome packaging. FHV and NoV are genetically only distantly related and are unable to replicate each other’s genome. The stringency of genome packaging was analyzed by studying the outcome of mixed infections in BHK cells. Immunofluorescence analysis of co-infected cells revealed that the coat proteins of the two viruses co-localize throughout the cytoplasm, indicating that FHV and NoV do not segregate into separate cellular locations for coat protein synthesis and virus assembly. Progeny virions, gradient-purified from co-infected cells, represented a mixture of NoV and FHV particles which showed typical nodaviral features by electron microscopy. RT-PCR analysis of RNA extracted from the particles confirmed that all four viral RNAs had been packaged into the viral progeny. FHV and NoV particles from co-infected cells separated by immunoprecipitation were also found to package all four viral RNAs indicating that these coat proteins can trans-encapsidate each other’s RNA. To further prove that NoV RNAs were packaged into particles assembled from FHV coat protein, Drosophila cells were infected with the progeny from mixed infections. Drosophila cells are susceptible to infection with particles assembled from FHV coat protein, but not NoV protein. Replicating NoV RNA1 and RNA2 were detected in infected Drosophila cells suggesting that they had been packaged into FHV particles. These results indicate that specific packa-ging of the nodavirus genome is not tightly controlled and unlikely based on sequence-specific encapsidation motifs.

113

A.18 Antigenic Switching of Hepatitis B Virus by Alternative

Dimerization of the Capsid Protein

Michael DiMattia1 1 National Institutes of Health Hepatitis B virus remains a major public health threat: 360 million people worldwide are chronically infected, resulting in one million deaths annually from liver cancer and cirrhosis. Two key aspects of HBV infection remain poorly understood: (1) how HBV evades the immune system in chronic infection and (2) the function of HBV e-antigen (eAg). Recent evidence suggests that the two may be related. eAg is a variant of the capsid protein (cAg; core-antigen) that does not assemble into capsids, is secreted into sera, and has different antibody reactivities than cAg. These observations are mysterious upon considering that eAg and cAg share the same protein sequence, save for 10 extra propeptide residues at the N-terminus of eAg. Nevertheless, eAg is used as a clinical marker for HBV replication and disease severity, and is present in all members of Hepadnaviridae, altogether suggesting it has an important and conserved function. We aimed to explore the long-standing question of how modest alteration of sequence termini can so profoundly affect the assembly and antigenic properties between cAg and eAg. To this end, we have crystallized and determined the structure of a complex of eAg and an anti-eAg monoclonal antibody fragment (Fab), exploiting a separately determined structure of the Fab. The antigens’ monomer folds are very similar; however in eAg, the propeptide induces a radically altered mode of dimerization (relative 140 deg. rotation) compared to cAg, locked into place through formation of a novel intramolecular disulfide bridge. To test the importance of the disulfide on eAg structure, negative-stain electron microscopy and analytical ultracentrifugation were used to assay eAg assembly state vs. oxidation state. Under reducing conditions, the eAg disulfide is disrupted and the subunits revert to a cAg-like association mode, forming capsid-like particles. The eAg structural switch reveals how capsid assembly is prevented and how a distinct antigenic repertoire is created, permitting evasion of the robust cytotoxic response that cAg evokes upon interaction with B cells. Despite this, the antigens remain highly cross-reactive at the T-cell level due to sequence identity. This duality is likely to be central to eAg’s role in establishing immune tolerance for cAg. The crystal structure now provides a framework upon which further study can fully elucidate the role of eAg in HBV persistence, both as an antigenic decoy and a direct modulator of the immune system.

114

B.1 Derepression and encapsidation of Staphylococcus aureus pathogenicity islands by helper phages

Rosie Hill1, Keith Manning1, Altaira D. Dearborn1, Erin A. Wall2, Gail E. Christie2 and Terje

Dokland1

1Dept. of Microbiology, University of Alabama at Birmingham, Birmingham, AL, USA 2Dept. of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements that encode virulence genes. SaPIs depend on a helper phage for their excision and encapsidation [1]. Without the helper phage, the SaPIs are maintained within S. aureus chromosome by the Stl global repressor. SaPI derepression is effected by specific phage proteins that disrupt the Stl-DNA interaction. Recently the phage 80α dUTPase (Dut) was reported as a derepressor of SaPIbov1 [2]. We previously showed that S. aureus Newman phage φNM1 is also able to derepress SaPIbov1[3] even though φNM1 Dut is highly divergent from 80α’s and is predicted to belong a different family of dUTPases. The objective of this study is to characterize the determinants of Dut derepression of SaPIbov1. As the first part of this work, we overexpressed and purified φNM1 Dut to determine its structure and possible interaction with SaPIbov1 Stl. Our hypothesis is that mobilization depends on a conserved GVSS motif in Dut.

[1] G.E. Christie and T. Dokland (2012) Pirates of the Caudovirales. Virology REF?? [2] M. A. Tormo-Mas, I. Mir, A. Shrestha, S. M. Tallent, S. Campoy, I. Lasa, J. Barbe, R. P. Novick, G. E.

Christie, and J. R. Penades. Nature. 465. 779-783 (2010) [3] A. Dearborn and T. Dokland. Bacteriophage. 2, 70-78 (2012)

115

B.2 Perspectives on the Crystal Structure of Human Adenovirus

Nhung Huynh1, Glen R. Nemerow2 and Vijay S. Reddy1

1 Department of Integrative Structural and Computational Biology, 2 Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

Replication defective and conditionally replicating adenoviruses (AdV) vectors are currently being utilized in ~25% of human gene transfer clinical trials. Rational development of adenovirus vectors for therapeutic gene transfer is hampered by the lack of accurate structural information. In particular, there exists a significant ambiguity with regards to the identity and location of the four cement proteins (IIIa, VI, VIII and IX) that stabilize the contiguous capsid shell and their role in mediating the interactions between the major capsid proteins. The recently determined X-ray structure of a human adenovirus (HAdV) vector at near atomic resolution represents a milestone as the largest biomolecular structure yet determined using X-ray diffraction methods[1]. The crystal structure revealed detailed interactions between the major capsid proteins and characteristic structural features of several accessory molecules that stabilize the AdV capsids. Recently, the refined crystal structure of HAdV has provided greater structural insights, which facilitated the revision of the previous assignments of several cement proteins derived from the cryoEM studies. The details of these new findings will be discussed. These new details unveiled by the refined crystal structure of HAdV represents a significant step forward in understanding the structural underpinnings of AdV assembly and cell entry mechanisms of a large dsDNA virus and provides new opportunities for improving adenovirus mediated gene transfer.

[1] Reddy VS, Natchiar SK, Stewart PL, Nemerow GR. Crystal structure of human adenovirus at 3.5 A resolution. Science. 2010;329(5995):1071-5.

116

B.3 Pressure-dependent genome ejections of double-stranded DNA viruses

Cathy Yan Jin1, J. A. Dover2, K. N. Parent2, W. M. Gelbart1, C. M. Knobler1

1 Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90024, USA 2 Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA

Double-stranded DNA viruses are highly pressurized. This high pressure provides a force for DNA ejection into host cells. In these in vitro studies we use bacteriophages lambda and P22 as examples, showing how the length of DNA ejected from the capsid depends on the effective capsid pressure arising from osmolyte (high-molecular-weight polyethylene glycol (PEG)) and polyvalent cation (spermine (sp4+)) in the external solution. Previous in vitro studies with bacteriophage lambda showed that ejection of DNA from purified virus particles can be partially inhibited by introducing an external osmotic pressure with PEG– the higher the concentration in the solution, the less the DNA ejected and thus the longer the length left inside the capsid [1]. Similarly, the ejected length can also be reduced by adding sp4+, thereby decreasing the pressure inside the capsid that is mainly due to DNA self-repulsion. We quantitatively studied the lambda DNA ejections in the presence of different concentrations of sp4+ and found that it begins to inhibit ejection only when its concentration is increased beyond 0.1mM. After reaching this threshold, around 70% of DNA remains inside the capsid even in the absence of PEG. We have also applied the PEG osmotic-suppression system to P22, a virus similar to lambda in being a double-stranded DNA bacteriophage, but differing in that it contains a number of copies of each of three different proteins (E proteins) that are ejected along with the DNA [2]. We have found a similar decrease in ejected DNA length with increase in PEG pressure. Since the E proteins also occupy a significant part of the capsid volume, we plan to use mutant P22s lacking one or two types of E proteins to see whether the packaging of the DNA is affected. Finally, we will determine the sequence of E protein ejection by controlling the partial ejection of DNA. [1] A. Evilevitch, M. Castelnovo, C. M. Knobler and W. M. Gelbart. J. Phys. Chem. B, 108, 21,

6838 (2004) [2] S. R. Casjens, P.A. Thuman-Commike. Virology, 411, 2, 393 (2011)

117

B.4 CryoEM-based geometric and dynamic analysis of

Nudaurelia capensis ωωωω virus maturation reveals the energy landscape of particle transitions

J. Tang1, B.M. Kearney2, Q. Wang3, P.C. Doerschuk4, T. Baker1, J.E. Johnson2

1 Department of Chemistry and Biochemistry, and Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093. USA. 2 The Scripps Research Institute, 10550 N. Torrey Pines Road MB-31. La Jolla, CA 92037. USA 3 Electrical and Computer Engineering, Cornell University, NY, USA

4 Biomedical Engineering and Electrical and Computer Engineering, Cornell University, NY, USA Quasi-equivalent viruses infecting animals and bacteria require a maturation process in which particles transition from an initially assembled procapsid to the infectious virion. In a remarkably efficient manner using only one or two gene products the virus creates an energy landscape that makes this a spontaneous process, occurring at the appropriate time and place. Nudaurelia

capensis ω virus (NωV) is a T=4, eukaryotic, ssRNA virus that is highly accessible for maturation studies. NωV procapsids (480Å), a maturation intermediate (410Å) and mature virion (410Å) were purified and structures determined by cryoEM. Subunit environments in these particles were quantitatively analyzed by employing geometric evaluation of an atomic model fit to an 8Å cryoEM reconstruction of the procapsid (pH 7.6) and the X-ray model of the mature capsid state (pH 5.0). Superposition of the procapsid quasi-equivalent subunits by rotations, with axes passing through the center of the particle, showed an overall atom RMSD of 3Å. Thus, quasi-symmetry elements related subunits with nearly the same precision as icosahedral symmetry. Dynamic analysis of the procapsid images with a maximum likelihood variance (MLV) method revealed that the entire particle had nearly the same, moderately low, variance, consistent with the homogeneous subunit environments. The first maturation intermediate (a mutant that has not undergone autocatalytic cleavage) and the fully mature particle are structurally indistinguishable at pH 5 at 6Å resolution. Geometric analysis of these particles revealed break down of quasi-equivalence, as average atom RMSD values were 11.4Å. MLV analysis showed that the variance of the mature particle was, on average, one fourth of the intermediate demonstrating that cleavage is essential to achieve the stability necessary for particle survival.

118

B.5 Effect of Spermidine on Bacteriophage phi29 DNA Packaging

Nicholas A. Keller1, Paul Jardine2, Shelley Grimes2, Douglas E. Smith1

1 University of California, San Diego, Mail Code 0379, 9500 Gilman Drive, La Jolla, CA, 92093 USA 2 University of Minnesota, 18-264 Moos Tower, 515 Delaware St. SE, Minneapolis, MN 55455

Molecular motors drive genome packaging in many dsDNA viruses against high forces resisting DNA confinement [1-4]. Polyamines screen the DNA charge and can induce DNA condensation into spooled conformations similar to those proposed to occur in phage packaging [5, 6]. Addition of polyamines has been shown to reduce DNA ejection forces and might conversely be expected to reduce forces resisting packaging [6-8]. Here, we report optical tweezers measurements of single DNA molecule packaging in phage phi29 with spermidine. Adding a low amount of spermidine, below the threshold for condensation, increases packaging rate by ~20% on average, implying a decrease in force resisting DNA confinement. Adding higher spermidine, sufficient to condense DNA, causes highly variable packaging dynamics. About 40% of complexes package faster (~25% faster, on average). Unexpectedly, however, the remainder exhibited dramatic slowing with frequent pausing and slipping, often before reaching 50% packaging. This inhibition is not due to a direct effect of spermidine on the motor or to condensation of unpackaged DNA. Rather, our findings suggest that the packaged DNA frequently forms unfavorable conformations which impede the motor. [1] Gelbart WM & Knobler CM. Science 323(5922): 1682-1683 (2009) [2] Smith DE. Current Opinion in Virology 1: 134 (2011) [3] Fuller DN, et al. Proc Natl Acad Sci U S A 104(27): 11245-11250 (2007) [4] Smith DE, et al. Nature 413(6857): 748-752 (2001) [5] Bloomfield VA. Biopolymers 44(3): 269-282 (1997) [6] Kindt J, Tzlil S, Ben-Shaul A & Gelbart WM. Proc Natl Acad Sci U S A 98(24): 13671-13674 (2001) [7] Evilevitch A, Lavelle L, Knobler CM, Raspaud E & Gelbart WM. Proc Natl Acad Sci U S A 100(16):

9292-9295 (2003) [8] De Frutos M, Brasiles S, Tavares P & Raspaud E. The European Physical Journal E 17(4): 429-434

(2005)

119

B.6 The structure of R135, a fiber associated enzyme of

Mimivirus

T. Klose1, D. Herbst2 and M.G. Rossmann1

1 Department of Biological Sciences, Purdue University, 240 S Martin Jischke Dr, West Lafayette, IN 47907 2 Biozentrum, University of Basel, Klingelbergstrasse 50 / 70, CH - 4056 Basel

Mimivirus, the prototypic member of the family of Mimiviridae is one of the largest viruses discovered so far. Its name is short for mimicking microbe, referring to the fact that the virus had initially mistakenly been identified as a microbe, mainly due to the fact that it stains Gram-positive. It was discovered later that this property is associated with a dense layer of 125nm long fibers that cover the virus with the exception of the star gate, a unique structure covering one of the fivefold vertices. The fibers are believed to play an important role during the infection process of the virus. Mimivirus seems to infect its host by being phagocytized and the virus likely benefits from a surface like that of Gram-positive bacteria, a common food source for amoeba. It has recently been determined that the fibers on the surface of the virus consist primarily of three proteins, R135, L725 and L829 [1].

Here we describe the structure of R135, a member of the Glucose/ Methanol/ Cholesterol (GMC)-oxidoreductase family. Members of this protein family are involved in capsid protein glycosylation [2]. Thus R135 is probably involved in modifying the fibers, possibly giving them their Gram-positive-like properties. Implications for the structure and potential function of the Mimivirus fibers will be discussed.

[1] M. Boyer, S. Azza, L. Barrassi, T. Klose, A. Campocasso, I. Pagnier, G. Fournous, A. Borg, C. Robert, X. Zhang, C. Desnues, B. Henrissat, M.G. Rossmann, B. La Scola, and D. Raoult. Proc. Natl. Acad. Sci. USA 108, 10296 (2011).

[2] M. Tonetti, D. Zanardi, J.R. Gurnon, F. Fruscione, A. Armirotti, G. Damonte, L. Sturla, A. de Flora, and J.L. van Etten. J. Biol. Chem. 278, 21559 (2003).

120

B.7 Insights into the Pseudomonas phage phi6 envelope assembly through expression of the component proteins in

Escherichia coli

O.L. Lyytinen1, C. Sèle1, L.P. Sarin1,2, J.J. Hirvonen1,2, P. Laurinmäki2, S.J. Butcher2, D.H.

Bamford1,2, M.M. Poranen1

1 Department of Biosciences, University of Helsinki, Viikki Biocenter, Helsinki, Finland 2 Institute of Biotechnology, University of Helsinki, Viikki Biocenter, Helsinki, Finland Pseudomonas phage phi6 is a well-known model organism for dsRNA virus assembly. Its virion consists of two structural layers assembled around an enzymatically active core termed the polymerase complex (proteins P1, P2, P4, and P7). This complex is responsible for the replication and transcription of the tri-segmented dsRNA genome. The polymerase complex is surrounded by the nucleocapsid surface shell (protein P8), which is crucial for penetrating the plasma membrane during phi6 entry into its host. The outermost virion layer is the envelope with host derived phospholipids and viral membrane proteins (P3, P6, P9, P10 and P13). Phi6 also encodes two non-structural proteins P12 and P14. Interestingly, P12 and the major envelope protein P9 are absolutely essential for virus-specific membrane formation to occur [1]. However, the mechanism of phi6 envelope formation and the role of non-structural protein P12 in this process are poorly understood. We have set up a production system for phi6-specific vesicles in Escherichia coli [2] and set out to characterize the vesicle formation pathway.

[1] M.D. Johnson 3rd, L. Mindich. J. Bacteriol. 176, 4124-4132 (1994) [2] L.P. Sarin, J.J. Hirvonen, P. Laurinmäki, S.J. Butcher, D.H. Bamford, M.M. Poranen. J. Virol. 86, 5376-5379 (2012)

121

B.8 Elucidating the Structure and Mechanism of Bacteriphage P22 Genome Packaging

R. McNulty1, A. Roy2, P. Prevelige3, G. Cingolani2, and J.E. Johnson1

1 The Scripps Research Institute, Department of Integrative Structural and Computational Biology,10550 North Torrey Pines Road, La Jolla, CA, USA 2 Thomas Jefferson University, Department of Biochemistry & Molecular Biology, 233 South 10th Street, Philadelphia, PA, USA 3 University of Alabama at Birmingham, Department of Microbiology, 845 19th St. South, Birmingham, AL, USA

The Salmonella-infecting bacteriophage P22 packages its genome from concatemers of dsDNA. This packaging machinery consists of a large (gp2) and small (gp3) terminase complex. Gp3 functions to properly position the gp2 for packaging initiation via the pac site [1]. Gp2 uses ATP hydrolysis to translocate a single copy of the genome into the bacteriophage procapsid. The motor activity of gp2 results in dsDNA packaging at a rate of up to 2000 bp/sec [2]. Although poorly understood, termination and cleavage are induced upon reaching a filled procapsid [3]. Upon cleavage, the gp2/3 complex quickly disassociates from the capsid enabling tail and associated proteins to bind, sealing the genome inside the procapsid. There is a crystal structure for the nanomeric gp3 [4,5] and only the nuclease domain of monomeric gp2 for P22 [6]. The goal is to clarify the oligomeric stoichiometry, orientation with respect to portal protein, and 3-D structure of gp2/3 complex using electron microscopy. Herein we present initial models of gp2/3 complex resulting from Random Conical Tilt TEM images. We seek to understand the molecular mechanism of genome packaging.

[1] Wu, H., Sampson, L., Parr, R. & Casjens, S. (2002). The DNA site utilized by bacteriophage P22 for initiation of DNA packaging. Mol Microbiol 45, 1631-46.

[2] Fuller, D. N., Raymer, D. M., Kottadiel, V. I., Rao, V. B. & Smith, D. E. (2007). Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability. Proc Natl Acad Sci U S A 104, 16868-73.

[3] Behnisch, W. & Schmieger, H. (1985). In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro. Virology 144, 310-7.

[4] Roy, A., Bhardwaj, A., Datta, P., Lander, G. C. & Cingolani, G. (2012). Small terminase couples viral DNA binding to genome-packaging ATPase activity. Structure 20, 1403-13.

[5] Nemecek, D., Lander, G. C., Johnson, J. E., Casjens, S. R. & Thomas, G. J., Jr. (2008). Assembly architecture and DNA binding of the bacteriophage P22 terminase small subunit. J Mol Biol 383, 494-501.

[6] Roy, A. & Cingolani, G. (2012). Structure of p22 headful packaging nuclease. J Biol Chem 287, 28196-205.

122

B.9 Mutations of putative Walker A and B motifs and Loop-Helix-Loop motif in large subunit of terminase

affect Phage Lambda DNA packaging

C-S Oh1, A. Ford1, M. Feiss1 1 Department of Microbiology, Carver College of Medicine, University of Iowa, 3-315 BSB, 51 Newton Rd. Iowa City, IA 52242. USA Bacteriophage lambda’s terminase plays an important role in DNA translocation and packaging during virus assembly. While the small subunit of terminase, gpNu1, contains the functional domains for dimerization and oligomerization, the large subunit, gpA, contains domains for translocation ATPase and packaging at the N-terminal and endonuclease at the C-terminal [1]. The sequence alignments of the N-terminal ATPase domain of phage lambda and other phage and viral terminases show four conserved motifs; Q, Walker A and B, and Coupling motifs [2]. In this study we focused on Walker A motif (76-KSARVGYS-83) which interacts with ATP, Walker B motif (174-VAGYD-178) which interacts with Mg-ATP complex, and E179 which is the proposed catalytic carboxylate. We also studied the structurally conserved loop-helix-loop region (191-GSPT-194) which has a putative role in motor velocity and processivity. Conserved residues in each motif were substituted to either amber codons which were crossed into phages and tested on various suppressors, or to various missense codons which were introduced into a plasmid containing gpA and tested by the complementation test. These mutants resulted in various phenotypes; lethal, reduced, or no effect to phage development. Some leaky mutants were then introduced into the expression vector and studied for gpA protein expression and its activity. [1] V. Rao, M. Feiss. Annu. Rev. Genet. 42:647-681 (2008) [2] B. Drapper, V. Rao. J. Mol. Biol. 369:79-94 (2007)

123

B.10 The cellular distribution of (+) RNA during Flock House virus infection: Implications for the selection of genomic

RNA during virus assembly

J.R. Short1, R. Gopal1 and A. Schneemann1

1 Department of Cell and Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA

The mechanisms involved in the specific packaging of multipartite genomes by (+) RNA viruses remain poorly understood. In order to gain insight into the location of genomic RNA encapsidation, we employed fluorescence in situ hybridization (FISH) to visualize the subcellular distribution of (+) vRNA in cells infected with the model nodavirus, Flock House virus (FHV). During FHV assembly, genomic RNAs 1 and 2 are encapsidated into a T = 3 icosahedral capsids which mature through autocatalytic cleavage of each capsid protein (CP) subunit to yield infectious virions. Although FHV virus-like particles readily package non-viral RNA in the absence of viral replication, over 98% of the RNA packaged during infection is genomic vRNA. Previously it has been shown that while vRNA replication and CP synthesis are initially targeted to different cellular locations (mitochondria and ER, respectively), these regions converge into perinuclear clusters late in infection. This suggested that the concurrence of CP with replicated (+) vRNA could facilitate a genome packaging mechanism involving specificity through proximity - the vast abundance of genomic RNA located where virus assembly is taking place ensures its preferential encapsidation. Our analysis of the cellular distribution of FHV RNA during infection, however, suggests that the vast majority of (+) RNA is trafficked towards the periphery of the cell relatively early during the infectious cycle (within 3 hours post infection) and does not relocate to the perinuclear region during mitochondrial condensation (15 hours post infection). In addition, the (+) RNA fits well within the region occupied by CP at the time points we tested. This suggests that genomic RNA is likely released from vRNA replication sites fairly soon after synthesis and trafficked to peripheral sites either during or prior to encapsidation. The role of both CP and B2 (a small virus-encoded protein known to bind dsRNA) in this process is currently under investigation.

124

B.11 Genetic Characterization of the Proposed Q and Coupling Motifs in the Packaging ATPase of Bacteriophage

Lambda

Jean Sippy1, Niky Vahanian1, Amir Sabbagh1, and Mike Feiss1

1 The University of Iowa Department of Microbiology, 51 Newton Road, Room 3-315 BSB, Iowa City, Iowa 52242 USA

We examined the residues which comprise two putative ATPase motifs in bacteriophage λ’s gpA: the coupling (C-motif), residues G212S213T214; and the Q motif, residues Y46Q47. The motifs became apparent after multiple alignment of translocases [1]. Various substitutions were tested with either amber codons which were crossed into phage and plated on various suppressor strains, or with missense codons, which were introduced into a complementation plasmid. The C-motif residues are stringent with respect to changes that yield functional terminase, and for the Q motif, we find that Y46 cannot be changed without loss of function. gpA’s critical Q residue does not, however, appear to be Q47 because λ growth is observed with several substitutions at this position.

[1] Draper and Rao JMB 369: 79-94 (2007).

125

B.12 Incorporation of portal protein in in vitro assembled P22 procapsids

Molly Siegel1, Juliana Cortines2, Ankoor Roy3, Gino Gingolani3, Carolyn Teschke1

1 University of Connecticut, Department of Molecular and Cell Biology, Storrs, CT 06269 2 Instituto de Microbiologia Paulo de Góes, Departamento de Virologia – UFRJ Edifício do Centro de Ciências da Saúde, Bloco I sala 55 subsolo Av. Carlos Chagas Filho , s/n Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ 3 Thomas Jefferson University, Dept. of Biochemistry & Molecular Biology, Philadelphia PA 19107

Incorporation of the portal protein complex is crucial to formation of mature infectious bacteriophage P22 because it is the location where the DNA is packaged into the procapsid. Packaging of DNA also triggers the maturation reaction. In vivo, the portal complex replaces a single penton in the icosahedral lattice. Conditions for in vitro incorporation of portal protein complexes during procapsid assembly have only been determined for phage phi29 and herpes virus. The mechanism by which the portal complex is incorporated into P22 procapsids is not understood. For instance, the rate of assembly of procapsids is the same whether the portal is present or not in infected cells [1]. This result indicates that portal protein may not be the nucleator of assembly of P22, unlike phage T4, where its portal has been demonstrated to be essential for proper nucleation. We have found conditions for incorporation of P22’s portal protein during in vitro assembly. Only monomeric portal protein is active for assembly; pre-assembled rings are not incorporated into the procapsids. Intriguingly, addition of portal monomers slows the assembly elongation time. The critical concentration does not appear to be affected by the addition of portal monomers. Our data suggest that we are altering the assembly nuclei leading to a decrease in the coat protein critical concentration [2].

[1] Bazinet, C. and J. King, J. Mol. Biol., 1988. 202(1): p. 77-86. [2] Katen, S. and A. Zlotnick, Methods Enzymol, 2009. 455: p. 395-417.

126

B.13 Testing the strength of adenovirus by single particle AFM nanoindentation

M.G.M. van Rosmalen1, K. Heinze1, C. Moyer2, G. Nemerow2, W. H. Roos1, G.J.L. Wuite1

1 Natuur- en Sterrenkunde, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, the Netherlands 2 Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA

Nanoindentation by atomic force microscopy (AFM) is an emergent technique to characterize the mechanical properties of nano-sized systems [1-2]. In addition, by using AFM one also obtains high-resolution topographical images of the sample. This allows both detailed morphological and mechanical analyses of single viral nanoparticles. Knowledge of morphology as well as mechanical properties is essential for understanding viral life cycles, including genome packaging, capsid maturation and uncoating.

A correlation between the elasticity of adenovirus with the early events on the virus life cycle was previously observed by Snijder et al. [3]. In this study, opposite effects on capsid stability where observed for binding of integrin, which facilitates virus endocytosis, and defensin, which restricts endosome escape and infection.

This current study includes three different types of mutants of the adenovirus. The mutants all have a single point mutation in an internal capsid protein, protein VI, that alters cell infectivity. Their morphology will be imaged by AFM and their mechanical properties characterized by nanoindentation. By correlating this to the ability of the mutants to enter host cells we obtain a clear insight on the link between mechanics and infectivity of adenovirus.

[1] J. Snijder, C. Uetrecht, R. Rose, R. Sanchez, G. Marti, J. Agirre, D. M. Guérin, G. J. Wuite, A. J. R. Heck, and W. H. Roos. Nature Chemistry 5, 502–509 (2013)

[2] W. H. Roos, R. Bruinsma, and G. J. L. Wuite. Nature Physics 6, 733-743 (2010) [3] J. Snijder, V. S. Reddy, E. R. May, W. H. Roos, G. R. Nemerow, and G. J. L. Wuite. J. Virol. 87, 2756

(2013)

127

B.14 Virion packaging and release constrain intrahost sequence diversity of influenza A virus

S.V. Venev*1, N. Renzette*2, P. Liu*3, D.R. Caffrey3, T.F. Kowalik2, C.A. Schiffer4, J.P. Wang3,

R.W. Finberg3 and K.B. Zeldovich1 1 Program in Bioinformatics and Integrative Biology, 2 Department of Microbiology and Physiological Systems, 3 Department of Medicine, 4 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA

Influenza A virus has a segmented -ssRNA genome and its virions are enveloped with the host cell derived lipid membrane. During early stages of infection, virions are released from an infected host cell leaving the cell membrane intact. It is well established that most influenza virions contain exactly 8 distinct RNA segments [1]. This specificity is presumably due to RNA-RNA interactions between viral RNA segments in the virion. Since the mutation rate of the influenza virus is high (~10-4 per nt per generation), we hypothesize that many influenza mutants fail to package in a virion and/or bud, and therefore remain in the infected host cell. As a consequence, genome packaging and virion budding may impose a constraint on intrahost sequence diversity of influenza virus. To test this hypothesis, we have grown A/Brisbane/59/2007 influenza virus in MDCK cell culture and used deep sequencing to compare SNP frequencies in cell associated viral RNA collected from cell lysate and in viral RNA from complete virions released into the supernatant. We found that sequence diversity of influenza virus RNA in the supernatant is markedly reduced compared to the cell associated viral RNA contained in the cell lysate. Frequencies of multiple individual SNPs in the influenza genome differ between the cell associated viral RNA and released virions. Analyzing the locations of SNPs affected by virion release, we found that a large fraction of such SNPs reside towards the 5’ and 3’ ends of influenza RNA segments, overlapping with previously established packaging signal regions. Analysis of enrichment of SNP frequencies in the virions compared to cell associated viral RNA unravels correlations between multiple sites in the genome, possibly explained by coevolution within RNA-RNA interaction motifs. These findings demonstrate that virion packaging and release impose sequence constraints on the influenza virus genome, and provide a novel method of identification of influenza packaging signals. The authors acknowledge the support of DARPA (Prophecy Program, Defense Sciences Office (DSO), Contract No. HR0011-11-C-0095) and the contributions of all the members of the ALiVE (Algorithms to Limit Viral Epidemics) working group. [1] Y. Chou, R. Vafabakhsh, S. Doğanay, Q. Gao, T. Ha, and P. Palese. PNAS 109 (23), 9101-9106 (2012)

128

B.15 Structural Studies of Bacteriophage T4 Baseplate by X-

ray Crystallography

M. L. Yap1, P. Plevka2, A. A. Aksyuk3, F. Arisaka4 and M. G. Rossmann.1

1 Department of Biological Sciences, Purdue University, West Lafayette, 47907 IN, USA. 2 CEITEC - Central European Institute of Technology, University Campus Bohunice, Kamenice 5/A4, CZ-62500 Brno, Czech Republic. 3 Laboratory of Structural Biology, NIH/NIAMS, Bethesda, MD, USA. 4 Grad Sch of Biosci and Biotech, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

The baseplate of bacteriophage T4 is a 6 MDa multiprotein complex comprised of 16 oligomeric proteins. Six wedges assemble around a central hub to form a baseplate. During infection, the baseplate changes its conformation from a dome to a hexagonal star-shaped structure. Structure of eight individual baseplate proteins had been determined previously by X-ray crystallography. The structure of the whole baseplate had been studied using cryo-electron microscopy (cryo-EM). CryoEM 3D reconstruction of the dome-shaped baseplate and the star-shaped baseplate with a contracted tail had been determined earlier to 12Å (1) and 17Å (2) resolution, respectively. By fitting X-ray crystal structures into the CryoEM density maps, it had been demonstrated how the baseplate rearrangement occurs during infection. However, molecular interactions among the proteins and conformational changes during infection are aspects that remain to be further clarified. Five recombinant wedge proteins (gp10, 7, 8, 6, and 53) were produced by using an E. coli expression system. The proteins self-assemble into wedges that further form into a hexagonal baseplate-like structure (3.3 MDa) resembling the contracted star-shaped baseplate but lacking the central hub (3). The protein complex was crystallized in 6% PEG 6000, 2M NaCl, Tris buffer at pH8. The crystals belong to the hexagonal space group P622 with unit cell dimensions a=b=470Å, c=457Å. The best crystals diffracted to 4.2Å resolution and a complete dataset was collected to 6Å resolution at the GM/CA-CAT beamline 23-ID-B of the Advanced Photon Source. Initial phases were generated by automated molecular replacement using partially solved protein structures as well as anomalous dispersion and isomorphous data using heavy atom thallium bromide clusters. Two wedges were positioned face to face in an asymmetric unit. Phases were improved by density modification by applications of solvent flattening, NCS averaging of the two wedges in an asymmetric unit and local symmetry averaging of individual oligomeric proteins. The interaction interfaces between proteins are to be explored.

[1] V. A. Kostyuchenko, P. G. Leiman, P. R. Chipman, S. Kanamaru, M. J. van Raaij, F. Arisaka. V. V. Mesyanzhinov, and M. G. Rossmann, Nat. Struct. Biol. 10, 688-693 (2003).

[2] P. G. Leiman, P. R. Chipman, V. A. Kostyuchenko, V. V. Mesyanzhinov, and M. G. Rossmann, Cell 118, 419-429 (2004).

[3] M. L. Yap, K. Mio, P. G. Leiman, S. Kanamaru, and F. Arisaka, J. Mol. Biol. 395(2), 349-360 (2010).

129

B.16 Molecular Simulation Study of the Gating Mechanism of the Lassa Virus Nucleoprotein

Jason Pattis1 and Eric R. May1

1 Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT

Lassa virus is the causative agent of Lassa fever, which infects several hundred thousand humans annually in Western African, resulting in greater than 5,000 deaths annually. The Lassa virus ambisense RNA genome encodes 4 proteins, the nucleoprotein (NP) is the most abundant in infected cells. The NP is involved in suppression of the innate immune system and interacts with the viral RNA to form the ribonucleoprotein complex in the complete virion. Multiple structures of the NP N-terminal domain [1,2] have recently been solved, but differences in the structure have led to different hypotheses about how the NP interacts with RNA. In one study it was proposed that the NP binds to mRNA cap [1]. While the other structure co-crystalized NP with a short ssRNA segment and based upon the structural differences between the two studies it was postulated that NP undergoes a gating mechanism, which allows it to bind viral RNA [2].

The primary focus of this investigation is to understand the mechanism by which the Lassa virus nucleoprotein (NP) binds to RNA and how conformational changes in the NP are achieved. Using conventional molecular dynamics, targeted molecular dynamics and umbrella sampling methods we are constructing pathways of the open-close transition of the NP in the presence and absence of an RNA substrate. From these studies we will gain insight into the thermodynamics of the process and be able to identify critical interactions in the pathway, which could then be targeted in mutational or drug discovery efforts.

[1] X. Qi, S. Lan, W. Wang, L.S. Schelde, H. Dong, G.D. Wallat, H. Ly, Y. Liang, and C. Dong. Nature 486, 779 (2010).

[2] K.M. Hastie, T. Liu, S. Li, L.B. King, N. Ngo, M.Z. Zandonatti, V.L. Woods, Jr., J.C. de la Torre, and E.O. Saphire. Proc. Natl. Acad. Sci. U.S.A., 108, 19365 (2011).

130

B.17 Characterization of ΦN - A fresh water bacteriophage

resembling cystoviruses

Sari Mäntynen1, Annika Kohvakka1, Elina Laanto1, Minna M. Poranen2, Jaana K.H. Bamford1, and Janne J. Ravantti2,3

1 Department of Biological and Environmental Science and Nanoscience Center, University of Jyväskylä, Finland 2 Department of Biosciences, University of Helsinki, Finland 3 Institute of Biotechnology, University of Helsinki, Finland Cystoviridae is a bacteriophage family, the type virus of which is Pseudomonas phage Φ6. Cystoviruses have three dsRNA genome segments enclosed in a polyhedral protein capsid which in turn is surrounded by a lipid-containing membrane. They have common structural and functional features with eukaryotic dsRNA viruses (e.g. reovirus, rotavirus, bluetongue virus and yeast L-A virus). Identified members of the Cystoviridae, all isolated from plant debris, infect gram-negative bacteria, primarily Pseudomonas syringae. We isolated and purified a fresh water bacteriophage ΦN which has striking similarities to the previously described cystoviruses. ΦN was isolated from a Pseudomonas sp. bacterium and purified. The bacteriophage lost infectivity in chloroform treatment indicating a lipid component in the structure. Electron microscopy of the ΦN virion revealed a membraneous structure surrounding a polyhedral head, resembling remarkably the structure of phage Φ6. Polyacrylamide gel electrophoresis analysis showed that the protein content of ΦN differs somewhat from that of Φ6. In addition, the sizes of the three RNA segments isolated from ΦN are close to, but not identical to, those of Φ6. Sequencing of the genome segments will be completed in near future. Interestingly, also the host range of the virus differs from Φ6 and other identified cystoviruses. Consequently, viral attachment and infection mechanism in general will be subjected to further investigation. The fresh water bacteriophage ΦN has evident similarities to the previously identified cystoviruses. Thus we postulate that it is a new member of this virus group. However, ΦN has also unique properties, e.g. divergent host range, giving interesting new insights into the Cystoviridae family.

131

B.18 Characterization of ΦN - A fresh water bacteriophage

resembling cystoviruses

Lu Xie1

1 Carnegie Mellon University This work will describe advances in learning rate parameters from assembly data to fit quantitative computer simulations to specific capsid assembly systems. We will examine the problem and our novel methods for learning parameters from bulk in vitro assembly data. We will then explore what the learned simulations can tell us about likely assembly pathways of these capsid systems. Finally, we will discuss extensions of this work to better predict how assembly might be altered in the cellular environment relative to bulk test tube assembly.

A starter’s guide for using

VIPERdb, a virus structure database

and a web portal for structural virology

Vijay Reddy and Jack Johnson

Integrative Structural and Computational Biology

The Scripps Research Institute,

La Jolla, CA 93027

URL: http://viperdb.scripps.edu

132

Contents

• Introduction to VIPERdb

• Home page

• Info_page

• Structure derived results

• Icosahedral server of quasi-equivalent lattices

• Gallery maker

• Virus origami: Fold-a-virus

• Navigating through VIPERdb

133

Introduction to VIPERdbVIPERdb (VIrus Particle ExploreR Database), http://viperdb.scripps.edu, is a relational database and a web portal for spherical virus structures determined by x-ray crystallography and cryoelectron microscopy and their structure derived properties. All the spherical viral capsids available in the PDB were transformed into a single icosahedral convention, Z(2),3,5,X(2), namely the VIPER convention for the ease of visualization and their computational analysis. Each entry in the database is analyzed in terms of subunit-subunit associations based on the extent of buried surface areas, identification of contacting residue residue pairs at the unique subunit interfaces and surface accessible residues. Each residue is annotated based on its location in the structure (e.g., surface, interface and core). All this information is stored in a relational MySQL database. Individual info_pages for each virus capsid are generated “on the fly” showing various pictorial representations of whole capsid and coat protein subunits and the information on the capsid size, T-number, net surface charge, taxonomy of the virus and a link to the primary citation. Various structure-derived properties/annotations are displayed as graphs, tables and interactive visual tools (e.g., JMOL, Google applets). All this information is grouped and represented in individual tabs (e.g., Biodata, Illustrations etc.,). Furthermore, a number of web-based utilities are provided to access/manipulate the structural information (e.g., oligomer generator, icosahedral server, galley maker, pdb2viper). As an outreach activity, we provide VIPER info_pages (password protected) of user submitted virus capsid coordinates to get access to the results of VIPER analysis prior to the publication of their structures. 134

VIPERdb homepagehttp://viperdb.scripps.edu

135

Contents of an info_pagehttp://viperdb.scripps.edu/info_page.php?VDB=1ohg

136

Some of the structure derived results available (can be accessed through annotations tab in the info_page)

1. Strong/weak locations in the

capsid

2. Surface accessible residues

3. Residues involved

in Crystal contacts

4. Similarity indices of interfaces

5. Lists of residue-pairs at the interfaces

137

Get access to the icosahedral cage of your

choice: http://viperdb.scripps.edu/icos_server.php?icspage=icos_gal

138

Make a portrait of virus capsids using the

gallery maker utility:http://viperdb.scripps.edu/gallery_maker.php

139

Virus Origami: Fold-a-virus

140

Navigating through VIPERdb with pull down menus

141

142

Previous Phage/Virus Assembly Meetings

Meeting Year Location Organizers I 1968 Lake Arrowhead, CA Bob Edgar, Bill Wood, Fred Eiserling II 1970 Povono, PA Lee Simon III 1972 Vail, CO Lloyd Kozloff, Don Cummings IV 1974 Squaw Valley, CA Fred Eiserling, Bill Wood, Sherwood Casjens V 1976 Snowbird, UT Sherwood Casjens VI 1978 Toronto, Ontario Helios Murialdo VII 1980 Asilomar, CA Rich Calendar VIII 1982 Fall Creek Falls, TN Gisela Mosig IX 1984 Navasota, TX Peter Berget X 1986 Montreal, Quebec Mike Dubow XI 1988 Asilomar, CA Fred Eiserling, Bob Duda XII 1991 Lakewood Cable, WI Dwight Anderson, Mike Feiss, Charlene Peterson XIII 1993 Syria, VA Lindsay Black, Bill Newcomb, Alasdair Steven XIV 1995 Johnston, PA Wade Gibson, Rodger Hendrix, Marjorie Russel XV 1997 Asilomar, CA Richard Calendar, David Coombs, Stan Person XVI 1999 Rio Rico, AZ Peter E. Prevelige, Ben Fane XVII 2001 Helsinki, Finland Dennis Bamford XVIII 2003 Woods Hole, MA Jonathan King XIX 2005 Winter Park, CO Carlos Catalano XX 2007 Toronto, Ontario Alan Davidson, Karen Maxwell XXI 2009 Veyrier-du-Lac, Annecy, France Paulo Tavares, Felix Rey XXII 2011 Port Aransas, TX Ian Molineux, Marc Morais, Ry Young

Previous FASEB Virus Assembly Conferences

Meeting Year Location Organizers I 1990 Saxtons River, VT Roger Burnett, Jon King, Peter Prevelige II 1992 Saxtons River, VT Roger Burnett III 1994 Saxtons River, VT Sherwood Casjens, Pat Spear IV 1996 Saxtons River, VT Roger Hendrix, Eric Hunter V 1998 Saxtons River, VT Sandra Weller, Alasdair Steven VI 2000 Saxtons River, VT Marie Chow, Mike Feiss VII 2002 Saxtons River, VT Jack Johnson, Margaret Kielian VIII 2004 Saxtons River, VT Lindsay Black, Peter Tatersall IX 2006 Saxtons River, VT Peter Prevelige, Anette Schneemann X 2008 Saxtons River, VT Robert Garcea, Bentley Fane XI 2010 Saxtons River, VT Marvis Agbandje-McKenna, Carlos Catalano XII 2012 Saxtons River, VT Rebecca Dutch, Venigalla Rao

XXIII Biennial Conference on Phage/Virus Assembly Participants

143

Nicola Gerardo Abrescia CIC bioGUNE, Spain Fred Antson University of York Nina Atanasova University of Helsinki Odisse Azizgolshani UCLA Dennis Bamford University of Helsinki Christian Beren UCLA Dave Bauer Carnegie Mellon University Zachary Berndsen UCSD Lindsey Black University of Maryland Brian Bothner Montana State University Devin Brandt UCLA Nina Broeker University of Potsdam, Germany Pascale Boulanger CNRS Patricia Campbell University of Pittsburg Sherwood Casjens University of Utah

Gino Cingolani Thomas Jefferson University James Conway University of Pittsburgh Nichole Cumby University of Toronto Li Dai Catholic University of America Wei Dai Baylor College of Medicine Alan Davidson University of Toronto Damian delToro UCSD Michael DiMattia NIH Terje Dokland University Alabama at Birmingham Tatiana Domitrovic TSRI Sarah Doore University of Arizona Bogdan Dragnea Indiana University Robert Duda University of Pittsburgh Eric Dykeman University of York Fred Eiserling UCLA


144

Kamel El Omari Strubi, Oxford, UK Alex Evilevitch Carnegie Mellon University Bentley Fane University of Arizona Michael Feiss University of Iowa Andrei Fokine Purdue University Chi-Yu Fu TSRI Song Gao The Catholic University of America Rees Garmann UCLA Peng Ge UCLA William Gelbart UCLA James Geraets University of York Preeti Gipson Baylor College Ramesh Goel University of Utah Boon Chong Goh University of Illinois, Urbana-Champaign Radhika Gopal TSRI

Paul Gottlieb City College of New York Sandra Greive University of York, UK Shelley Grimes University of Minnesota Cameron Haase-Pettingell MIT Michael Hagan Brandeis University Steve Harvey Georgia Tech Roger Hendrix University of Pittsburgh Rosie Hill University of Alabama, Birmingham Chuan Hong Baylor College of Medicine Alexis Huet University of Pittsburgh Nhung Huynh TSRI Paul Jardine University of Minnesota Wen Jiang Purdue University Cathy Yan Jin UCLA John E. Johnson TSRI


145

Shuji Kanamaru Tokyo Institute of Technology Al Katz City College of New York Bradley Kearney TSRI Nicholas Keller UCSD Jonathan King MIT Thomas Klose Purdue University William Klug UCLA Charles Knobler UCLA Abhay Kotecha University of Oxford, UK Shannon Kruse University of Washington Andreas Kuhn University of Hohenheim Amelie Leforestier CNRS, France Petr Leiman EPFKL, Switzerland Françoise Livolant CNRS, France Aida Llauró Portell University of Madrid, Spain

Juan Luis Loredo University of York, UK Outi Leena Lyytinen University of Helsinki Sari Mantynen University of Jyvaskyla, Finland Donna-Rae Marquez University of Arizona Karen Maxwell University of Toronto Eric May University of Connecticut Reginald McNulty TSRI Yulia Meshcheriakova John Innes Centre Ian J. Molineux University of Texas, Austin Marc Morais University of Texas, Galveston Tina Motwani University of Connecticut Bonnie Oh University of Pittsburgh ChoonSeok Oh University of Iowa Philomena Ostapchuk Stony Brook University Kristin Parent Michigan State University


146

Jason Perimutter Brandeis University Grigore Pintille Baylor College of Medicine Guido Polles SISSA, Italy Peter Prevelige University of Alabama, Birmingham Vijay Reddy TSRI Venigalia Rao Catholic University of American Mariska van Rosmalen Vrije University Amsterdam Michael Rossmann Purdue University Andrew Routh TSRI Aaron Roznowski University of Arizona Senjuti Saha University of Toronto Pooja Saxena John Innes Centre Susan Schroeder University of Oklahoma Philip Serwer University Texas Health Science Center James Short TSRI

Surendra W. Singaram UCLA Jean Sippy University of Iowa Doug Smith UCSD Tom Smith Danforth Plant Center Nicole Steinmetz Case Western Reserve University Alasdair Steven NIH Peter Stockley University of Leeds, UK Lei Sun Purdue University Paulo Tavares CNRS, France Carol Teschke University of Connecticut Denise Tremblay Universite Laval Guillaume Tresset University of Paris, France Roman Tuma University of Leeds, UK Marie-Christine Vaney Institut Pasteur, France David Veesler TSRI


147

Sergey Venev U Mass Medical School Lu Xie Carnegie Mellon University Moh Lan Yap Purdue University

Mark Young Montana State University Mohamed Zairi CNRS, France Zhengyi Zhao University of Kentucky

148

Acknowledgements The meeting would not have occurred without the dedicated effort of many individuals in addition to the chairs. Olive Ireland expertly managed the registration procedure and kept track of the financial records. Grants and contracts at Scripps, lead by Vice President Kaye Wynne, provided the resources to handle credit card processing and account maintenance and management. David Veesler, Tatiana Domitrovic, Chi-Yu Fu, Andrew Routh, Reginald McNulty and Bradley Kearney drafted the program that was then further refined by the chairs. Tatiana and Nathalia Domitrovic created the cover art for the abstract book with help from Reginald McNulty. Tatiana Domitrovic and Hanna Miller designed the T-shirts. Bradley Kearney devoted dozens of hours to create the final program and abstract book, and arrange for its printing. His facility with spreadsheets was essential to keep track of all aspects of the meeting organization. His expertise was crucial for the success of the enterprise and he deserves special recognition for his tireless and good-natured effort when things were stressful. Finally we thank our sponsors for their generous support of the meeting.

149

Sponsors

The Agouron Institute

The Scripps Research Institute

Elsevier - Virology

Documents

Table of Contents - Scripps Research Institutepva.scripps.edu/files/pdf/PVA2013.pdf · Table of Contents Table of Contents ... Architecture of a viral capsid in complex with the maturation