II Reunión

17 - 19 de abril, 2013

Facultad de Ciencias

Módulo C0

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PROGRAM

Wednesday, April 17th

08:45-9:00 Opening 9:00-10:00 S. Blanc (UMR-BGPI Montpellier, France) " Gene copy number is differentially regulated in a multipartite

virus " Page 10

10:00-10:30 Santiago F. Elena (Instituto de Biología

Molecular y Celular de Plantas (CSIC-UPV), Campus UPV

València) " Empirical fitness landscapes reveals a limited number of

accessible adaptive pathways for an RNA virus" Page 11 10:30-11:00 Ester Lázaro (Centro de Astrobiología (CSIC-

INTA) " Genetic and Phenotypic Properties of Bacteriophage Qβ

Populations Evolved at Increased Error Rate" Page 12

11:00-11:30 COFFEE 11:30-12:00 José A. Cuesta (Universidad Carlos III) " Evolving on Phenotype Landscapes" Page 14 12:00-12:30 José J. Ramasco (Instituto de Física

Interdisciplinar y de los Sistemas Complejos IFISC (CSIC-

UIB)) "Real-time numerical forecasts of global epidemic spreading"

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Page 15 12:30-13:00 Vicente Pallàs (Instituto de Biología Molecular y

Celular de Plantas – CSIC) " Presentación del Grupo de Virología Molecular de Plantas

del IBMCP”Page 16 13:00-15:00 LUNCH 15:00-15:30 Carlos Briones (Centro de Astrobiología (CSIC-

INTA)) " Magnesium-Dependent RNA Folding of the Internal

Ribosome Entry Site of Hepatitis C Virus Genome Monitored

by Atomic Force Microscopy " Page 20 15:30-16:00 Mauricio G. Mateu (Centro de Biología

Molecular “Severo Ochoa”) " Manipulation and Biological Implications of the Thermal

Stability and Mechanical Properties of Viruses”Page 22 16:00-16:30 Carmen San Martin (Centro Nacional de

Biotecnología, CSIC) " Structural Determinants of Adenovirus Assembly” Page 23

16:30-17:00 COFFEE 17:00-17:30 P. J. de Pablo (Unversidad Autónoma de Madrid) "Physical virology with Atomic Force Microscopy” Page 24

17:30-18:00 D.M.A. Guérin (Unidad de Biofísica (CSIC-

UPV/EHU) - Fundación Biofísica Bizkaia) " Triatoma Virus (TrV) capsid disassembly and genome

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release" Page 25 18:00-18:30 Teresa Ruiz-Herrero (Universidad Autónoma de

Madrid) " Dynamic simulations of virus budding” Page 27

Thursday, April 18th

09:00-10:00 Félix Rey (Institut Pasteur) " Class II viral membrane fusion proteins: virus/host gene

exchanges and cell-cell fusion events in multicellular

organisms" Page 28 10:00-10:30 Núria Verdaguer (Institut de Biologia Molecular

de Barcelona CSIC) " Structural Characterization of RNA Viruses" Page 29

10:30-11:00 Nicola Abrescia (Structural Biology Unit, CIC

bioGUNE, CIBERehd) " Three-dimensional Visualization of Forming Hepatitis C

Virus-like Particles by Electron-Tomography" Page 30 11:00-11:30 COFFEE 11:30-12:00 J.R. Castón (Centro Nacional de Biotecnología

CSIC) "Cryo-EM structure of Penicillium chrysogenum virus at 4 Å

resolution" Page 32 12:00-12:30 Daniel Luque (Instituto de Salud Carlos III)

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"Structural Analysis of Rotavirus Infection Associated

Macromolecular Complexes " Page 33 12:30-13:00 Carmela Garcia-Doval (Centro Nacional de

Biotecnología CSIC) " Structural biology of viral fibres " Page 34 13:00-15:00 LUNCH 15:00-15:30 José L. Carrascosa (Centro Nacional de

Biotecnología CSIC) "Studies on the double stranded DNA packaging machinery of

viruses” Page 36 15:30-16:30 Roundtable 16:30-17:00 COFFEE 17:00-18:30 Posters session

21:00 Gala dinner. Restaurant “Gasset 75”

Friday, April 19th

09:00-10:00 Antonio Šiber (Institute of Physics, Croatia) "Are electrostatic and elastic properties of viruses tuned by

evolution and how?" Page 38

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10:00-10:30 J. Hernández-Rojas (Universidad de la Laguna) "A minimalist potential energy model for the self-assembly of

virus capsids" Page 39 10:30-11:00 David Reguera (Universitat de Barcelona)

“Physical Modeling of the Self-Assembly and Mechanical

Properties of Viruses” Page 40

11:00-11:30 COFFEE 11:30-12:00 A.M. Bittner (CIC nanoGUNE) "The Physics of Tobacco Mosaic Virus" Page 41 12:00-12:30 Andres de la Escosura (Universidad Autónoma

de Madrid) " Self-Assembly Triggered by Self-Assembly: Virus-Like

Particles Loaded with Supramo-lecular Nanomaterials" Page

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12:30-13:00 A. Velazquez-Campoy (Universidad de

Zaragoza) " NS3 Protease from Hepatitis C Virus: Biophysical

Characterization of a Partially Disordered Protein Domain"

Page 45

13:00-13:15 Closure

13:15 LUNCH

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ORAL PRESENTATIONS

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Gene copy number is differentially regulated in

a multipartite virus

Stéphane Blanc

UMR-BGPI Montpellier, France

Multipartite viruses are enigmatic entities with a

genome divided into several nucleic acid segments, each

encapsidated separately. An evident cost for these viral

systems, highly enhanced if some segments are rare, is the

difficulty to gather at least one copy of each segment to ensure

infection. We tackle the question of the segment frequency-

related cost by monitoring the relative copy number of the 8

single-gene segments composing the genome of a plant

nanovirus during host infection. We show that some viral genes

accumulate at very low frequency, whereas others dominate.

We further show that the relative frequency of viral genes

impacts on both viral accumulation and symptom expression,

and specifically changes in different hosts. All earlier proposed

benefits of viral genome segmentation do not depend on the

frequency of the segments and cannot explain the situation

described here. We propose that the differential control of gene

(or segment) copy number may provide a major unforeseen

benefit for multipartite viruses, which may compensate for the

extra-costs related to the existence of low-frequency segments.

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Empirical fitness landscapes reveals a limited

number of accessible adaptive pathways for an

RNA virus

Santiago F. Elenaa,b

, Francisca de la Iglesiaa, and Jasna

Lalića

aInstituto de Biología Molecular y Celular de Plantas (CSIC-UPV),

Campus UPV CPI 8E, Ingeniero Fausto Elio s/n, 46022 València,

Spain. bThe Santa Fe Institute, 1399 Hyde Road Park, Santa Fe, NM 87501,

USA

RNA viruses are the main source of emerging

infectious diseases owed to the evolutionary potential bestow

by their fast replication, large population sizes and high

mutation and recombination rates. However, an equally

important parameter, which is usually neglected, is the

topography of the fitness landscape, that is, how many fitness

maxima exist and how well connected they are, which

determines the number of accessible evolutionary pathways.

To address this question, we have reconstructed a fitness

landscape describing the adaptation of Tobacco etch potyvirus

(TEV) to a new host, Arabidopsis thaliana. Two fitness traits

were measured for most of the genotypes in the landscape,

infectivity and virus accumulation. We found prevailing

epistatic effects between mutations in the early steps of

adaptation, while independent effects became more common at

latter stages. Results suggest that the landscape was highly

rugged, with a reduce number of potential neutral paths and a

alternative fitness peaks, being the one reached by the evolving

TEV population not the global optima.

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Genetic and Phenotypic Properties of

Bacteriophage Qβ Populations Evolved at

Increased Error Rate

Ester Lázaroa, Laura Cabanillas

a, and María Arribas

a

aCentro de Astrobiología (CSIC-INTA), Ctra de Ajalvir Km 4,

Torrejón de Ardoz, 28850 Madrid (Spain)

RNA virus replication takes place at a very high error

rate. When the number of mutations per genome increases

about a certain value, mutation can outpace selection, causing

fitness decreases, and sometimes population extinction.

Most mutations decrease the thermodynamic and kinetic

folding stability of proteins, reducing their capability to

perform optimally. Since virus capsids are the result of the

correct assembly of multiple copies of one or several proteins,

it would be expected that mutations leading to incorrect

foldings also reduced capsid stability, which could result in a

higher sensitivity to adverse environmental conditions.

One of the projects carried out in our laboratory is focused on

the study of the genetic and phenotypic characteristics of virus

populations evolved at increased error rate. Our experimental

model is the bacteriophage Qβ propagated in the presence of a

mutagenic nucleoside analogue (5-azacytidine or AZC). The

phenotypic traits we have evaluated include the replicative

ability of individual viruses and their thermal stability at

temperatures above 50º C. Our results show that hypermutated

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viruses are more prone to lose their infectivity upon exposition

at high temperatures. However, the repetition of a few cycles

of exposition to this adverse condition, followed of replication

of the surviving viruses, leads to the selection of virus

populations with increased stability in hot environments.

The genetic analysis of the populations evolved at increased

error rate has allowed us to investigate how beneficial

mutations that reduce the sensitivity to AZC spread at

increased error rate. We have found that the process is affected

by interference between different mutations and also by

antagonistic epistasis, resulting in the prolonged permanence of

polymorphisms [1,2]. We have also found that the main

mutation conferring AZC resistance is located in a protein

which is present at low amount in the capsid. The result

suggests that the correct assembly of the virus capsid is one of

the main targets of selection in the presence of AZC.

1. M. Arribas, L. Cabanillas and E. Lázaro, Virology 417, 343-352

(2011). 2. L. Cabanillas, M. Arribas, and E. Lázaro, BMC Evolutionary

Biology 13:11 (2013).

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Evolving on Phenotype Landscapes

José A. Cuestaa,b

and Susanna C. Manrubiaa,c

aGrupo Interdisciplinar de Sistemas Complejos (GISC),

Departamento de Matemáticas, Universidad Carlos III de Madrid,

Avda. de la Universidad 30, 28911 Leganés, Madrid, Spain bInstituto de Biocomputación y Física de Sistemas Complejos (BIFI),

Universidad de Zaragoza, 50009 Zaragoza, Spain cCentro de Astrobiología, CSIC-INTA, Carretera de Ajalvir km 4,

28850 Torrejón de Ardoz, Madrid, Spain

Despite the usefulness of genotype landscapes they are

a source of potential misunderstandings. The reason is that the

genotype-to-phenotype map is highly degenerated. Huge

patches of the genotype landscape (genotype networks, GN)

correspond to just a single phenotype. Natural selection is blind

to genotypic differences within the same GN. As genotype

landscapes are patchworks of GN, populations traversing them

by accumulating mutations will exhibit quite an unusual

dynamic behavior. We develop a simplified model of

phenotype landscape inspired by quantitative studies of GN of

RNA. As a first approximation, this landscape can be regarded

as a network of interconnected phenotypes. Individuals with

the same phenotype reproduce at the same rate. Populations

jump from a given phenotype to a neighboring one, but the rate

at which they do is determined by topological properties of the

GN, in particular its size and the time already spent within it.

This renders the evolutionary process non-Markovian. We

explore the implications of this phenotype-based evolutionary

model for the adaptability of quasi-species as well as for

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phylogeny.

Real-time numerical forecasts of global

epidemic spreading

José J. Ramascoa and the GLEaM team

aInstituto de Física Interdisciplinar y de los Sistemas Complejos

IFISC (CSIC-UIB), Campus UIB, 07122 Palma, Spain .

Mathematical and computational models for infectious

diseases are increasingly used to support public-health

decisions. Their capacity to forecast disease arrival times,

number of cases or even the quantities of drugs or beds needed

to treat patients could suppose a major leap forward for doctors

and health-system managers. However, the reliability of these

methods to offer good quality predictions must be proven. Data

gathered for the 2009 H1N1 influenza crisis represent an

unprecedented opportunity to validate real-time model

predictions and define the main success criteria for different

approaches. We used the Global Epidemic and Mobility Model

to generate stochastic simulations of epidemic spread

worldwide, yielding (among other measures) the incidence and

seeding events at a daily resolution for 3,362 subpopulations in

220 countries. Using a Monte Carlo Maximum Likelihood

analysis, the model provided an estimate of the seasonal

transmission potential through the Monte Carlo likelihood

analysis and generated ensemble forecasts for the activity

peaks in the northern hemisphere in the fall/winter wave. These

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results were validated against the real-life surveillance data

collected in 48 countries, and their robustness assessed by

focusing on 1) the peak timing of the pandemic; 2) the level of

spatial resolution allowed by the model; and 3) the clinical

attack rate and the effectiveness of the vaccine.

M. Tizzoni et al., BMC Medicine 10, 165 (2012).

Presentación del Grupo de Virología Molecular

de Plantas del IBMCP

Vicente Pallàs

Instituto de Biología Molecular y Celular de Plantas (IBMCP) (UPV-

CSIC); Av. De los Naranjos S/N; Ed. 8E, 46022 Valencia.

The main objectives that the Plant Molecular Virology

Group of the IBMCP addresses are the following: (1) Intra-

and inter-cellular movement of viruses and viroids in their

susceptible host plants; (2). Protein and RNA trafficking trough

vascular tissues and (3). Characterization of host factors

interacting with viral genes that are responsible of the viral

susceptibility and/or resistance. To address these objectives we

use three different RNA pathogens: Carmovirus,

Alfamo/Ilarvirus, and viroids.

Carmoviruses are one group of viruses with the almost

simplest genome organization known. Melon necrotic spot

carmovirus (MNSV) is a small (~30 nm), isometric plant virus

that has an icosahedral symmetry with a triangulation number

of T=3. Virions are composed of 180 identical CP subunits,

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which consist of three subunits (A, B and C). The CP is

divided into three domains, designated as the RNA-binding

domain (R), the shell domain (S), and the protruding domain

(P). The arm region is located between the R and S domains,

and the hinge region is between the P and S domains. The P

domain projects outward from the virus particle and has a

characteristic anti-parallel β-sheet called a jellyroll

conformation, which has been found in a variety of proteins

having ligand binding functions. The MNSV genome consists

of a 4.3-kb, positive-sense, ssRNA containing five ORFs,

including p29, p89, p7A, p7B and p42. The coat protein (CP)

is encoded on p42. We have demonstrated that p7A and 7B are

involved in virus movement. P7A has RNA-binding properties

being this activity essential for the cell to cell movement

(Navarro et al., 2006; Genovés et al., 2009). P7B is an integral

membrane protein harbouring a unique transmembrane domain

which is also essential for intracellular movement (Martinez-

Gil et al., 2007: Genovés et al., 2011). We have demonstrated

that an active COPII-dependent early secretory pathway is

required for the intra- and intercellular cell-to-cell movement

of MNSV, revealing the involvement of the Golgi apparatus in

this process (Genovés et al., 2010). Alfalfa mosaic virus (AMV), a member of the

Bromoviridae family of plant viruses, occurs predominantly as

bacilliform particles with a diameter of 19 nm and a length

varying from 30 to 56 nm composed of one of the four

genomic RNAs and a surrounding shell built from a single

gene product of 220 residues. AMV particles are labile

structures held together predominantly by RNA-protein

interactions. The RNA is exposed at the outside and the CP

assembles into T = 1 spheres of 60 subunits. In addition to

virion formation, the coat protein (CP) of AMV is involved in

the regulation of replication and translation of viral RNAs, and

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in cell-to-cell and systemic movement of the virus. An

intriguing feature of the AMV CP is its nuclear and nucleolar

accumulation. We have recently identified an N-terminal

lysine-rich nucleolar localization signal (NoLS) in the AMV

CP required to both enter the nucleus and accumulate in the

nucleolus of infected cells, and a C-terminal leucine-rich

domain which might function as a nuclear export signal (NES)

(Herranz et al., 2012). Moreover, we demonstrated that AMV

CP interacts with importin-α, a component of the classical

nuclear import pathway. A mutant AMV RNA 3 unable to

target the nucleolus exhibited reduced plus-strand RNA

synthesis and cell-to-cell spread. Moreover, virion formation

and systemic movement were completely abolished in plants

infected with this mutant. In vitro analysis demonstrated that

specific lysine residues within the NoLS are also involved in

modulating CP-RNA binding and CP dimerization, suggesting

that the NoLS represents a multifunctional domain within the

AMV CP. The observation that nuclear and nucleolar import

signals mask RNA-binding properties of AMV CP, essential

for viral replication and translation, supports a model in which

viral expression is carefully modulated by a cytoplasmic/

nuclear balance of CP accumulation.

Navarro JA, Genoves A, J. Climent, A. Sauri, L. Martinez-Gil, I.

Mingarro and Pallas V. (2006) RNA-binding properties and

membrane insertion of Melon necrotic spot virus (MNSV)

double gene block movement proteins. Virology 356: 57-67. Martinez-Gil, L., Sauri, A., Vilar, M., Pallas, V. and Mingarro, I.

(2007). Membrane insertion and topology of the p7B

movement protein of Melon necrotic spot virus (MNSV).

Virology 367 (2): 348-357. Martínez-Gil, L., Sanchez-Navarro, J.A., Cruz, A., Pallas, V., Perez-

Gil, J., Mingarro, I. (2009). Plant Virus Cell-to-Cell

Movement Is Not Dependent on the Transmembrane

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Disposition of Its Movement Protein. J. Virol. 83(11): 5535-

5543. Genovés A, Navarro JA and Pallas V. (2009). A self-interacting

carmovirus movement protein plays a role in binding of viral

RNA during the cell-to-cell movement and shows an actin

cytoskeleton dependent location in cell periphery. Virology

395: 133-142. Genovés A, Navarro JA and Pallas V. (2010). The Intra- and

Intercellular movement of Melon necrotic spot virus

(MNSV) depends on an active secretory pathway. Molecular

Plant Microbe Interactions 23(3): 263-272. Genovés, A., Pallás, V. and Navarro, J.A. (2011).Contribution of

topology determinants of a viral movement protein on its

membrane association, intracellular traffic and viral cell-to-

cell movement. J. Virol. 85(15): 7797-7809. Herranz, M.C., Pallas V, Aparicio F. (2012). Multifunctional roles for

the N-terminal basic motif of Alfalfa mosaic virus coat

protein: nucleolar/cytoplasmic shuttling, modulation of

RNA-binding activity and virion formation. Mol. Plant

Microbe Interact. 25(8): 1093-1103.

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Magnesium-Dependent RNA Folding of the

Internal Ribosome Entry Site of Hepatitis C

Virus Genome

Monitored by Atomic Force Microscopy.

Ana García-Sacristán

a,b, Elena López-Camacho

a,c, Ascensión

Ariza-Mateosb,d

, Miguel Moreno

a, Rosa M. Jáudenes

a, Jordi Gómez

b,d, José

Ángel Martín-Gagoa,c

and Carlos Briones

a,b.

a Department of Molecular Evolution, Centro de Astrobiología

(CSIC-INTA), Torrejón de Ardoz, Madrid. b Centro de Investigación Biomédica en Red de enfermedades

hepáticas y digestivas. (CIBERehd), Spain. c Instituto de Ciencia de Materiales de Madrid (CSIC), Cantoblanco,

Madrid. d Instituto de Parasitología y Biomedicina “López-Neyra” (CSIC),

Granada.

The 5’ untranslatable region (5’UTR) of the hepatitis C

virus (HCV) genomic RNA is highly structured and contains

an internal ribosome entry site (IRES) element responsible to

drive cap-independent translation initiation (1). The ion-

dependent tertiary fold of the minimal HCV IRES element

(containing domains II to IV) has been investigated (2), and

significant progress has been made in determining the three-

dimensional structure of individual IRES domains and

subdomains at high resolution (3). Nevertheless, little

information is still available (4) on the tertiary structure of the

HCV IRES element.

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Atomic Force Microscopy (AFM) is a useful

nanotechnology-based tool for the analysis of a wide range of

biological entities, including nucleic acids and their complexes

(5). We have optimized AFM technology for analysing HCV

IRES structure in native conditions as well as for monitoring

its conformational rearrangements in diverse physicochemical

environments, in particular at magnesium ion concentrations

ranging from 0 to 10 mM. Here we report the Mg2+

-dependent

folding of the HCV IRES in a sequence context that includes

its structured, functionally relevant flanking regions (domains

I, V and VI). In the 571 nt-long HCV genomic RNA molecule

analyzed, a structural switch has been monitored when Mg2+

concentration increases from 2 to 4 mM. This effect has been

confirmed by classical molecular biology techniques for RNA

structural characterization, such as gel-shift analysis and partial

RNase T1 cleavage. Our results suggest a magnesium-driven

transition from an ‘open’ to a relatively ‘closed’ conformation

of the HCV IRES.

1. P. J. Lukavsky. Virus Res. 139: 166 (2009) 2. J. S. Kieft et al. J. Mol. Biol. 292: 513 (1999) 3. K. E. Berry et al. Structure 19: 1456 (2011) 4. J. Pérard et al. Nat Commun. 4: 1612 (2013) 5. H. G. Hansma et al. Curr. Op. Struct. Biol. 14: 380 (2004)

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Manipulation and Biological Implications of the

Thermal Stability and Mechanical Properties of

Viruses

Mauricio G. Mateu

Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM),

Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid

We are using protein engineering to manipulate the

thermal stability and mechanical properties of small viruses.

We aim at understanding the molecular determinants that

underlie the physical properties of viruses, and also at the

design of viral particles with improved thermal and/or

mechanical resistance for bio/nanotechnological applications.

We have recently discovered that electrostatic repulsions

between subunits in the capsid of foot-and-mouth disease virus

(FMDV) underlie its low thermostability, and we have

engineered thermostable FMDV variants for improved

vaccines. In collaboration with Dr. P.J.de Pablo´s and

J.Gómez´s groups (Dept. of Physics of the Condensed Matter,

UAM) we found that, in the minute virus of mice (MVM),

segments of the viral DNA bound to specific sites in the capsid

act like molecular buttresses that decrease the mechanical

elasticity of most regions in the viral particle. However, the

regions around channels involved in biologically relevant

molecular translocation events are kept free from bound DNA,

and remain as elastic as in the empty capsid. Our recent studies

using atomic force microscopy indicate that this anisotropic

distribution of mechanical stiffness may be a biological

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adaptation to prevent MVM inactivation without impairing

infection. We have also mechanically disassembled single

MVM particles using AFM, and experimentally identified

theoretically predicted assembly/disassembly intermediates.

Finally, these studies have led to the engineering of the

mechanically stiffer viral capsids known to date.

Structural Determinants of Adenovirus Assembly

Carmen San Martín

Department of Macromolecular Structure. Centro Nacional de

Biotecnología (CNB-CSIC). Darwin 3, 28049 Madrid (Spain)

We focus on the principles governing assembly and

stabilization of complex viruses, using adenovirus (AdV) as a

model system. The dsDNA AdV genome is bound to large

amounts of positively charged proteins that help condense it

forming the core, which is confined inside a T=25 icosahedral

capsid composed by multiple copies of seven different viral

proteins. The final stage of AdV morphogenesis consists in

proteolytic processing of several capsid and core proteins. The

immature virus, containing all precursor proteins, is not

infectious due to an uncoating defect. To determine why the

presence of precursor proteins impairs uncoating, we have

carried out in vitro disruption analyses of mature and immature

capsids. The results show how maturation primes the virus for

stepwise uncoating in the cell, and reveal the structural changes

undergone by the virion in conditions similar to those

24

encountered during entry(1)

. We have also contributed to define

the role of the viral genome as a cofactor of the AdV protease

during maturation(2,3)

. Current research interests include the

mechanism of genome packaging and the organization of the

non-icosahedral components in the virion. 1. A. J. Pérez-Berná et al., J Biol Chem 287, 31582 (2012). 2. V. Graziano et al., J Biol Chem 288, 2068 (2013). 3. P. C. Blainey et al., J Biol Chem 288, 2092 (2013).

Physical virology with Atomic Force Microscopy

Pedro J. de Pablo Gómez

Universidad Autónoma de Madrid, 28049 Madrid, Spain.

Viruses are striking examples of macromolecular

assembly of proteins, nucleic acids, and sometimes lipid

envelopes that form symmetric objects with sizes ranging from

10s to 100s of nanometers. The basic common architecture of a

virus consists of the capsid a protein shell made up of repeating

protein subunits, which packs within it the viral genome which

can be single or double stranded DNA or RNA depending on

the type of the virus. Virtually every aspect of the virus cycle

from DNA packing to maturation to interaction with the host

modifies and, in turn, is influenced by the material properties

of the virus. In this talk I will show how Atomic Force

Microscopy has emerged as a unique technique to unveil some

25

physical properties of viruses, such as stiffness and elasticity,

which can be directly related to their structure and function (1).

In addition, AFM enables monitoring the dynamics of virus

disassembly in real time to unveil the ultimate physical

changes to trigger virus infectivity (2).

1. Hernando-Pérez, M., Miranda, R., Aznar, M., Carrascosa, J. L.,

Schaap, I. A. T., Reguera, D., and de Pablo, P. J. Small 8, 2365

(2012). 2. Ortega-Esteban, A., Pérez-Berná, A. J., Menéndez-Conejero, R.,

Flint, S. J., San Martín, C., and de Pablo, P. J. Scientific Reports 3,

1434 (2013).

Triatoma Virus (TrV) capsid disassembly and

genome release

Rubén Sánchez-Eugenia and Diego M.A. Guérin

Unidad de Biofísica (CSIC-UPV/EHU), and §Fundación Biofísica

Bizkaia. Bº Sarriena S/N, 48940 Leioa, Bizkaia, Spain. Email:

diego.guerin@ehu.es

TrV is a small spherical, non-enveloped, +ssRNA virus

that infects triatomines (Hemiptera: Reduviidae), and belongs

to the Dicistroviridae family (1). Dynamic Light Scattering and

intrinsic fluorescence experiments at low pH (<5.0) indicate

that acidification does not affect capsid integrity(2). Cryo-EM

3D reconstructions show that, after genome release, the

26

resulting empty capsid do not displays striking conformational

changes in reference to the native viral capsid (3). Atomic

Force Microscopy nanoindentation and Native Mass

Spectrometry experiments show that the encapsidated RNA

plays an important role in stabilizing the viral integrity, and

that the interplay between protein shell and genome is highly

dependent on the pH (4). These and other experiments,

demonstrate that in TrV, the genome release displays features

that are in contrast with the current model of genome delivery

based on the mammalian viruses poliovirus and rhinovirus. 1) "Characterization of Triatoma virus, a Picorna-like virus

isolated from the Triatomine bug Triatoma infestans". O. A.

Muscio, J. L. La Torre, E. A. Scodeller (1988). J. Gen. Virol. 69

:2929–2934. 2) "Capsid protein identification and analysis of Triatoma Virus

(TrV) mature virions and naturally occurring empty particles".

Agirre, J., Aloria, K., Arizmendi, J.M., Iloro, I., Elortza, F., Marti,

G.A., Neumann, E., Rey, F.A., and Guérin, D.M.A. (2011) Virology

409:91-101. 3) “Cryo-TEM reconstruction of Triatoma virus particles: a clue

to unravel genome delivery and capsid disassembly”. Agirre, J.,

Goret, G., LeGoff, M., Sánchez-Eugenia, R., Marti, G.A., Navaza, J.,

Guérin D.M.A., and Neumann, E. (2013) J. Gen. Virol. In press

(DOI: 10.1099/vir.0.048553-0). 4) "Probing the biophysical interplay between a viral genome

and its capsid" J. Snijder, C. Uetrecht, R.J. Rose, R. Sanchez-

Eugenia, G.A. Marti, J. Agirre, D.M.A. Guérin, G.J.L. Wuite, A.J.R.

Heck, W.H. Roos (2013). Nature Chemistry. In press.

27

Dynamic simulations of virus budding

Teresa Ruiz-Herreroa and Michael Hagan

b

aDepartamento de Física Teórica de la Materia Condensada,

Universidad Autónoma de Madrid. b Department of Physics, Brandeis University, Waltham, MA, USA

For many viruses assembly and budding occur

simultaneously during the last stage of the replication cycle.

Understanding the basic mechanisms of this process could

promote biomedical efforts to block viral replication and

enable use of capsids in nanomaterials applications. To this

end, we have performed molecular dynamics simulations on a

coarse-grained model to elucidate the special characteristics for

virus assembly on a fluctuating surface. Our simulations show

that the membrane promotes assembly through dimensional

reduction of adsorbed subunits, but also introduces barriers that

inhibit complete assembly. We find that a domain within the

membrane (i.e. lipid raft) 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.

28

Class II viral membrane fusion proteins:

virus/host gene exchanges and cell-cell fusion

events in multicellular organisms

Félix Rey

Institut Pasteur / CNRS

Paris, France Class II proteins are viral membrane fusogenic molecules

folded essentially as β-sheet and having an internal fusion

peptide. In particular, they lack the characteristic central alpha-

helical coiled coil present in the post-fusion conformation of

all other viral fusion proteins. The regular, icosahedrally

symmetric enveloped viruses that have been studied so far,

such as flaviviruses, alphaviruses and phleboviruses have been

shown to have class II fusion proteins, which in their pre-

fusion conformation make an icosahedral shell surrounding the

viral membrane. Yet despite having very similar envelope

proteins, these viruses belong to three different viral families

with totally different genome replication machineries. We have

recently identified the rubella virus fusion a belonging to class

II, although the virus particles appear pleomorphic and lack

icosahedral symmetry. In spite of the lack of any detectable

sequence conservation, the available structures indicate that

class II proteins have undergone divergent evolution from a

distal, ancestral gene. We have now discovered that the cellular

fusion protein EFF-1, involved in syncytium formation during

the genesis of the skin in nematodes (C. elegans) and in other

multicellular organisms, is also folded as a class II viral fusion

protein, thereby indicating common ancestry, highlighting an

unprecedented amount of exchange of genetic information

29

between viruses and cells. My talk will discuss the

implications of this finding, which highlights the intricate

exchange of genetic information that has taken place between

viruses and cells during evolution. This analysis also suggests a

mechanism for the homotypic cell-cell fusion process, which

has not been studied so far.

Structural Characterization of RNA Viruses

Núria Verdaguer

Institut de Biologia Molecular de Barcelona CSIC, Parc

Científic de Barcelona Baldiri i Reixac10, 08028, Spain.

The replicative cycle in RNA viruses relies on: i) the

attachment to the appropriate cellular receptors, efficient entry

into the host cell and delivery of the viral RNA into the

cytoplasmon, ii) the activity of unique virus-encoded enzymes,

leading to viral RNA and protein synthesis and, iii) the

assembly of infectious virions that are released from the cell to

continue the infectious process. The structural and non-

structural viral proteins that orchestrate these steps are

potentially vulnerable targets for “attack” by appropriate

ligands that interfere with their functionality. Results of our recent research, aimed at the elucidation of

the X-ray structures of different viral proteins and protein-RNA

complex assemblies involved in RNA uncoating and RNA-

dependent RNA replication, will be presented.

30

Three-dimensional Visualization of Forming

Hepatitis C Virus-like Particles by Electron-

Tomography

Daniel Badia-Martinez

a, Bibiana Peralta

a, German Andrés

b,

Milagros Guerrab, David Gil-Cartón

a and Nicola Abrescia

a,c

aStructural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio,

Spain. bCentro de Biología Molecular Severo Ochoa, CSIC-UAM, Campus

Cantoblanco, 28049 Madrid, Spain.

cIKERBASQUE, Basque Foundation for Science, 48011 Bilbao,

Spain.

Hepatitis C virus (HCV) infects almost 170 million people per

year being one of the major causes for chronic liver disease. As

other flaviviruses, HCV is thought to replicate in the cytoplasm

acquiring the viral envelope by budding through the endoplas-

matic reticulum (ER) but its assembly pathway with the in-

volvement of lipiddroplets, architecture and structures of its

envelope proteins are poorly understood. With this paucity of

three-dimensional (3D) structural information, applying a re-

ductionist and mechanistic approach we embarked in studying

HC virus-like particles produced in insect cells. Using electron

tomography of plastic-embedded sections of Sf9 cells, we have

provided a 3D morphological description of these HCV-LPs at

the ER site as surrogate of wt-HCV allowing to view the parti-

cles one-by-one and each in its budding stage (differently to

the previously used 2D imaging technique that displays the

HCV-LPs as projection and whose shape doesn’t necessarily

reflect the budding stage). Tomographic data were collected on

our JEOL JEM2200-FS microscope on a 4Kx4K CCD camera.

31

Tomograms were processed with IMOD, denoised using To-

mobflow and analysed with Chimera and Amira softwares. Our

data provide a 3D sketch of viral assembly at the ER site with

different budding stages identified as three main classes: (i)

membrane areas of protein concentration, (ii) cup-shaped parti-

cles and (iii) particles on the verge of scission. Furthermore we

could detect proximity of buds from which we hypothesize a

mechanism of large particles formation.

Acknowledgments

We are extremely grateful to Genentech and S. Foung for providing

respectively the AP33 antibody and the

antibodies CBH-2, -5 and -7 against glycoprotein E2.

32

Cryo-EM structure of Penicillium chrysogenum

virus at 4 Å resolution

J.R. Castóna, J. Gómez-Blanco

a, D. Luque

a, D. Garriga

b, José

M. Gonzáleza, A. Brilot

c, W.H. Havens

d, J.L. Carrascosa

a, B.L.

Truse, N. Verdaguer

b, N. Grigorieff

c and S.A. Ghabrial

d

aCentro Nacional de Biotecnología/CSIC, 28049 Madrid, Spain;

bIBMB/CSIC, 08028 Barcelona, Spain;

cBrandeis University,

Waltham MA, USA; dUniversity of Kentucky, Lexington KY, USA;

eCIT-NIH, Bethesda MD, USA

Penicillium chrysogenum virus (PcV) is a fungal

dsRNA virus with a genome comprised of four segments. The

PcV capsid is based on a T=1 lattice formed by 60 subunits.

Whereas the PcV capsid protein (CP) has two motifs with a

similar fold, most dsRNA virus capsid subunits consist of

dimers of a single protein (with a 120-subunit capsid). This

ubiquitous stoichiometry provides an optimal framework for

genome replication and organization. We report the 3D structure by single-particle cryo-EM analysis

of PcV at ~4 Å resolution. The full-atom model of the 982-

amino-acid CP showed the critical contacts among subunits

that mediate capsid assembly, and specific RNA-protein

interactions. Despite the lack of sequence similarity between

the two halves, the CP is an almost perfect structural

duplication of a single domain in which most -helices and -

chains matched very well. Superimposition of secondary

structure elements showed a single “hot spot” into which

structural and functional variations can be introduced by

insertion of distinct segments.

33

The near-atomic structure of the PcV capsid protein derived

from cryo-EM data has allowed us to determine that its

conserved core is a hallmark fold preserved in the dsRNA virus

lineage.

Structural Analysis of Rotavirus Infection

Associated Macromolecular Complexes

Daniel Luquea, Esther Martín-Forero

a, Fernando González-

Camachoa, María del Carmen Terrón

a, José L. Carrascosa

b,

José R. Castónb, Javier M. Rodríguez

a

a CNM-ISCIII. Carretera de Majadahonda - Pozuelo, Km. 2.200.

28220 - Majadahonda (Madrid). bCNB-CSIC. C/ Darwin nº 3, Cantoblanco. 28049 Madrid.

Our laboratory combines electron microscopy and image

processing methods with molecular biology techniques in order

to determine structure-function relationships in medically

important human pathogens. One of our main research topics is

Rotavirus, the most relevant member of the family Reoviridae

due to its public health significance and its role as a model in

the research of this complex family of dsRNA viruses.

To become fully infectious, the rotavirus virion spike

protein must be proteolytically cleaved by trypsin-like

proteases in the intestinal lumen. To investigate the mechanism

underlying this step we have analyzed cleaved and intact

rotavirus particles by cryo-electron microscopy. These studies

have revealed a new trypsin-independent reorganization of the

34

virus spike and its importance for virus infectivity.

Unlike the rest of the Reoviridae, whose morphogenesis is

purely cytoplasmic, the immature rotavirus particles enter the

reticulum, are surrounded by a membrane, lose that membrane

and are released into the cytoplasm as mature. We have

addressed the production and structural characterization of this

rotavirus Membrane Enveloped Particles (MEPs) to understand

the rotavirus morphogenesis and as a model for the study of the

transport of protein complexes across the endoplasmic

reticulum.

Structural biology of viral fibres

Carmela Garcia-Dovala, Laura Córdoba García

a, Meritxell

Granell Puiga, Abhimanyu K. Singh

a, Thanh H. Nguyen

a,

Marta Sanz Gaiteroa, Mark J. van Raaija

aDepartamento de Estructura de Macromoléculas, Centro Nacional

de Biotecnología (CNB-CSIC) c/Darwin, 3 28049 Madrid

Our research focuses on the fibres some viruses use to

attach to their host cells. These fibres have a common

structure: a N-terminal virus attachment domain, a shaft

domain and a C-terminal receptor-binding domain involved in

host recognition. Our goal is to determine the structure of these

fibres and determine their role in host recognition.

During the last two years we solved the structures of the C-

terminal head domains of two different adenovirus fibres from

35

hitherto unknown genera: one from the Turkey type 3

Siadenovirus and one from the Snake type 1 Atadenovirus.

In 2010, we published the structure of the C-terminal

domain of gp37 (1), the distal half of bacteriophage T4 fibre.

Now we have also solved the structure of part of the proximal

fibre protein gp34 and in collaboration with the group of Pedro

de Pablo (UAM) we are performing AFM experiments on full-

length gp37.

We also solved the structure of the C-terminal domain of the

bacteriophage T7 fibre (2) and we are doing a mutational

analysis of the residues that may be involved in receptor

recognition.

1. Bartual, S. G., Otero, J. M., Garcia-Doval, C., Llamas-Saiz, A. L.,

Kahn, R., Fox, G. C. and van Raaij, M. J. Proc Natl Acad Sci U S A

107, 20287-20292 (2010). 2. Garcia-Doval, C. and van Raaij, M. J. Proc Natl Acad Sci U S A

109, 9390-9395 (2012).

36

Studies on the double stranded DNA packaging

machinery of viruses.

José L. Carrascosaa,c

Rebeca Bocanegraa, A. Cuervo

a and M.

Ibarraa

aCentro Nacional de Biotecnología, CSIC, c/Darwin 3, Cantoblanco,

28049 Madrid, Spain

cInstituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA

Nanociencia), Cantoblanco, 28049 Madrid, Spain

Double stranded DNA bacteriophages (dsDNA)

package actively their genetic material inside the capsid using

a protein motor which requires the hydrolysis of ATP as energy

source. DNA enters inside the viral head through the channel

formed by the connector that sits at a unique five-fold vertex of

the icosahedral capsid, a vertex which is also involved in the

delivery of the genome during DNA ejection.

We are studying the different components of this

machinery, namely the connector (which forms the

dodecameric portal at the viral vertex), the terminase (which is

a powerful motor that converts ATP hydrolysis into mechanical

movement of the DNA) and the tail proteins (which prevent

DNA exit and, upon a signal, promote the ejection of the DNA

from the viral head).

We have determined that the terminase assembled into

the portal complex shows a different conformation when

compared to the isolated terminase pentamer. The function

of the portal vertex is studied by efficient orthogonal

integration of the connector into lipid bilayers that allows to

37

perform DNA packaging in liposomes with integrated

connectors. We use a new experimental set up based in the

combination of patch clamp and optical tweezers. We are also

working in the structure of the DNA ejection machinery. We

have determined the precise topology of the tail structural

proteins by comparing the structure of the T7 tail extracted

from viruses and a recombinant complex formed by gp8, gp11

and gp12, and our high resolution model reveals the existence

of a common architecture with other Podoviridae tail

complexes.

38

Are electrostatic and elastic properties of viruses

tuned by evolution and how?

Antonio Šiber

Institute of physics, Bijenička c. 46, 10000 Zagreb, Croatia

Viruses have been studied as electrostatic and

elastic entities, yet, not much is really known regarding

the possible evolutionary convergence of their

electrostatic and elastic properties. Are there physical

constraints and reasons for these properties to converge at

all? Can we possibly say something regarding the

evolutionary relatedness of different viruses on the basis

of their physical properties? I will present some results

from my studies of elastic [1-3] and electrostatic [4-8]

properties of viruses and discuss the constraints that these

properties impose on a functional, evolutionary viable

virus.

[1] A. Šiber, "Buckling transition in icosahedral shells subjected to

volume conservation constraint and pressure: Relations to virus

maturation", Phys. Rev. E 73, 061915 (2006). [2] A. Šiber and R. Podgornik, "Stability of elastic icosadeltahedral

shells under uniform external pressure: Application to viruses under

osmotic pressure“, Phys. Rev. E 79, 011919 (2009). [3] A. Lošdorfer Božič, A. Šiber and R. Podgornik, “Statistical

analysis of sizes and shapes of virus capsids and their resulting

elastic properties”, in press in J. Biol. Phys., DOI 10.1007/s10867-

013-9302-3 [4] A. Šiber and R. Podgornik, "Role of electrostatic interactions in

39

the assembly of empty spherical viral capsids", Phys. Rev. E 76,

061906 (2007). [5] A. Šiber and R. Podgornik, "Nonspecific interactions in

spontaneous assembly of empty versus functional single-stranded

RNA viruses“, Phys. Rev. E 78, 051915 (2008). [6] A. Šiber and A. Majdandžić, "Spontaneous curvature as a

regulator of the size of virus capsids“, Phys. Rev. E 80, 021910

(2009). [7] A. Lošdorfer Božič, A. Šiber, and R. Podgornik, "How simple can

a model of an empty viral capsid be? Charge distributions in viral

capsids“, J. Biol. Phys. 38, 657 (2012). [8] A. Šiber, A. Lošdorfer Božič, and R. Podgornik, "Energies and

pressures in viruses: contribution of nonspecific electrostatic

interactions“, Phys.Chem.Chem.Phys. 14, 3746 (2012).

A minimalist potential energy model for the self-

assembly of virus capsids

J. Hernández-Rojas, J. Bretón, and J.M. Gomez Llorente

Departamento de Física Fundamental II and IUdEA,

Universidad de La Laguna, 38205, Tenerife, Spain

We present a simple potential energy model built as a sum

of pair-wise anisotropic interactions for viruses with two types

of capsomers: pentamers and hexamers. While the pentamer-

hexamer interaction parameters depend on the number of

capsomers, the hexamer-hexamer potential parameters are the

same for all capsids. “Basin-hopping” global optimization

method is used to find the lowest energy structures for virus

40

capsids with up to N=176 (Bacteriophage T4 head). Among

these structures we find those of the icosahedral viruses. We

also find prolate and oblate capsids based on Moody’s rules

and other new structures derived from the hexagonal lattice.

Physical Modeling of the Self-Assembly and

Mechanical Properties of Viruses

David Reguera

Departament de Física Fonamental, Universitat de Barcelona, C/

Martí i Franquès 1, 08028 Barcelona, SPAIN

Viruses are fascinating biological entities, in the fuzzy

frontier between life and inert matter. Contrary to most

biological organisms, viral particles are made of a minimal

number of relatively simple components that are not capable of

any metabolic activity, except when their genome sequesters

the metabolism of the infected host to achieve the replication

of new particles. Despite the lack of sophisticated biological

machinery, viruses have found the way to efficiently infect the

host, assemble, and egress the cell following, in many cases, a

coordinated sequence of passive and spontaneous processes.

This strongly suggests that, during their life cycle, viruses must

rely on general physical and chemical mechanisms to succeed

in their different tasks and to achieve the required resistance

against possible extreme environmental conditions.

In this talk, I will summarize some of our recent efforts

41

to understand the basic physical principles behind the

virus life-cycle. In particular, I will focus on the kinetics

and thermodynamics of assembly of empty capsids using

computer simulations of simplified coarse-grained

models. We will also discuss how these models can be

very useful to understand the remarkable mechanical

properties of the resulting capsid. The results of these

studies provide new insights into the microscopic

mechanisms of the assembly process and the physical

ingredients controlling the selection of a particular

structure that can be potentially very useful to develop

biomedical and nanotechnological applications.

The Physics of Tobacco Mosaic Virus

A.M. Bittnera,b

aCIC nanoGUNE, San Sebastián, Spain.

bIkerbasuqe, Bilbao, Spain.

How do nanoscale fibres (and tubes!) interact with water

(and with other liquids)? The answer has to rely on a very good

choice of the fibre (or tube). The use of plant viruses is

motivated by their simple structure, well-defined diameter, and

well-characterised chemical behaviour.

The Self-Assembly group employs scanning probe and

environmental electron microscopy techniques to Tobacco

42

mosaic virus. We observe wetting scenarios below 50 nm.

However, the big challenge is the molecular scale below 5 nm.

We were able to address it indirectly with TEM: We try to

clarify how solutions of metal complexes interact with the

virus, with a special focus on effusion from the 4 nm channel

inside the virion. Potential uses of plant viruses include acting

as templates for nanoscale materials, and as drug delivery

vehicle.

1. J.M. Alonso et al., review Trends in Biotechnol., subm. (2013). 2. A.A. Khan et al. Langmuir 29 (2013) 2094-2098. 3. J.M. Alonso et al. Nanotechnol. 24 103405 (2013). 4. S. Balci et al., Nanotechnol., 23 045603 (2012). 5. A. Mueller et al. ACS Nano, 5 (2011) 4512-4520. 6. A. Kadri et al. Virus Res., 157 (2011) 35-46.

43

Self-Assembly Triggered by Self-Assembly:

Virus-Like Particles Loaded with Supramo-

lecular Nanomaterials

Andres de la Escosura,a Melanie Brasch,

b Jealemy

Galindo,b Eduardo Anaya,

a Francesca Setaro,

a Daniel

Luque,c Jose L. Carrascosa,

c Jose R. Caston,

c Jeroen J. L.

M. Cornelissenb and Tomas Torres

a,d

aUniversidad Autónoma de Madrid, Organic Chemistry Department,

Cantoblanco, 28049 Madrid (Spain) fLaboratory for Biomolecular Nanotechnology, MESA+ Institute,

University of Twente, PO Box 207, 7500 AE Enschede (The

Netherlands) cDepartment of Structure of Macromolecules, Centro Nacional de

Biotecnología/CSIC, Cantoblanco, 28049 Madrid (Spain) dIMDEA-Nanociencia, Facultad de Ciencias, Ciudad Universitaria

de Cantoblanco, 28049 Madrid (Spain)

The self-assembly of biomolecules such as the coat proteins

(CP) of virus capsids offer great opportunities in

nanotechnology and nanomedicine, leading to monodisperse

platforms where different chemical species can be organized

through covalent or non-covalent bonding. Yet, because the

covalent approach for the modification of virus capsids is still a

demanding task, efficient and straightforward supramolecular

strategies are highly desirable. The Cowpea Chlorotic Mottle

Virus (CCMV), in particular, is a plant virus of 28 nm in

diameter with an interesting sensitivity to pH and ionic

strength. Depending on these factors, CCMV capsids can

rapidly be disassembled in vitro into CP dimers and then re-

assembled again. In this presentation, we will show several

44

examples of hierarchical and cooperative processes in which

self-assembled organic chromophores serve as templates for

the assembly of the CCMV CP around them. In such processes,

the structure of the self-assembled templates determines the

size and geometry of the resulting virus-like particles (VLP),

while confinement within the VLP also determines the

structure of the chromophore self-assemblies. The precise

structure and assembly properties of these particles have been

studied in detail by microscopy techniques, and sophisticated

VLP have been designed and prepared for multimodal

photodynamic therapy (PDT) and imaging.

“Viruses and Protein Cages as Nanocontainers and Nanoreactors”,

A. de la Escosura, R. Nolte and J. Cornelissen, J. Mat. Chem. 2009,

19, 2274-2278.

“Encapsulation of DNA-Templated Chromophore Assemblies within

Virus Protein Nanotubes”, A. de la Escosura, P. Janssen, A.

Schenning, R. Nolte and J. Cornelissen, Angew. Chem. Int. Ed. 2010,

49, 5463-5466.

“Encapsulation of Phthalocyanine Supramolecular Stacks into Virus-

Like Particles”, M. Brasch, A. de la Escosura, Y. Ma, A. Heck, T.

Torres and J. Cornelissen, J. Am. Chem. Soc. 2011, 133, 6881.

“Self-Assembly Triggered by Self-Assembly: Protein Cage

Encapsulated Micelles as MRI Contrast Agents ”, J. Galindo, M.

Brasch, E. Anaya, A. de la Escosura, et al. Submitted.

“Structure and Assembly Properties of Phthalocyanine-Loaded Virus-

Like Particles”, D. Luque, A. de la Escosura, et al. Manuscript in

preparation.

45

NS3 Protease from Hepatitis C Virus:

Biophysical Characterization of a Partially

Disordered Protein Domain

O. Abiana,b

, S. Vegaa, C. Marcuello

c, A. Lostao

c,d, J.L.

Neiraa,e

, A. Velazquez-Campoya,d,f

a Institute for Biocomputation and Physics of Complex Systems

(BIFI), Joint Unit BIFI-IQFR (CSIC), Universidad de Zaragoza,

Zaragoza, Spain b Centro de Investigación Biomédica en Red en el Área Temática de

Enfermedades Hepáticas y Digestivas (CIBERehd), ISCIII; Aragon

Health Sciences Institute (I+CS) – IIS Aragon, Zaragoza, Spain c Laboratorio de Microscopías Avanzadas (LMA), Instituto de

Nanociencia de Aragón (INA), Universidad de Zaragoza, Spain d ARAID Foundation, Government of Aragon, Spain

e Instituto de Biología Molecular y Celular, Universidad Miguel

Hernández, Elche (Alicante), Spain f Department of Biochemistry and Cellular and Molecular Biology,

Faculty of Sciences, Universidad de Zaragoza, Zaragoza, Spain

The NS3 protease from the hepatitis C virus is located at the

N-terminal domain of the non-structural protein 3. It has been

considered as a drug target since its identification as a key

enzyme in the viral life cycle. A biophysical characterization

performed on this protein has unraveled a quite complex

conformational landscape for this allosteric enzyme, with a

substantial interplay between its intrinsic plasticity and the

interactions with cofactors (zinc and viral protein NS4A) and

substrates (1-3).

46

1. X. Arias-Moreno, O. Abian, S. Vega, J. Sancho and A.

Velazquez-Campoy. Curr. Protein Pept. Sci. 12, 325-338 (2011). 2. O. Abian, S. Vega, J. L. Neira and A. Velazquez-Campoy.

Biophys. J. 99, 3811-3820 (2010). 3. O. Abian, J. L. Neira and A. Velazquez-Campoy. Proteins 77,

624-636 (2009).

47

POSTERS

Aznar P1 .......................................................................................................... 48 Bocanegra P2 .................................................................................................. 50 Carrillo, P.J.P P3.............................................................................................. 51 Condezo P4 ..................................................................................................... 53 Correia P5 ........................................................................................................ 54 Cuervo A P6 .................................................................................................... 56 Ferrero D P7 .................................................................................................... 57 Guérin P8......................................................................................................... 59 Hernando-Pérez P9 ......................................................................................... 60 Iranzo P10 ....................................................................................................... 62 Llauró P11 ....................................................................................................... 64 Mertens P12 .................................................................................................... 65 Ortega-Esteban P13 ........................................................................................ 66 Pérez-Berná AJ P14 ....................................................................................... 67 Rincón V P15 .................................................................................................. 69 Rodrigo P16 .................................................................................................... 71

48

P1. Physical ingredients controlling the viral

capsid

María Aznar and David Reguera

Universidad de Barcelona,

Dpto. Física Fundamental, Martí i Franqués, 1,

08028 Barcelona

Spain.

Email: maznar@correu.ffn.ub.es.

One of the crucial steps in the viral life cycle is precisely the

self-assembly of its protein shell. Typically, each native virus

self-assembles into a unique T- number structure, with some

exceptions like Hepatitis B Virus, which makes T=3 and T=4

capsids. But many viruses have the capability to self-assemble

into different T-number and shape structures in vitro by

changing the assembly conditions (i.e. typically the pH, salt

and protein concentrations). For example, Polyoma [1] or

Simian Virus 40 [2] self-assemble in vitro into T=1, snub

cubes, T=7 and different size tubes.

A proper understanding of the ingredients that control the in

vitro assembly of viruses is essential to get capsids with well-

defined size and structure that could be used for promising

applications in medicine or bionanotechnology. However, the

mechanisms that determine which of the possible capsid shapes

and structures is selected by a virus and that avoid its

polymorphism are still not well known.

We present a coarse-grained model to analyze and understand

the physical mechanisms controlling the size and structure

49

selection in viral self-assembly [3]. We have characterized the

phase diagram and the stability of T = 1, 3, 4, 7 and snub cube

structures using Monte Carlo simulations. In addition, we have

studied the tolerance of the different shells to changes in

physical parameters related to ambient conditions. Finally, we

will discuss the factors that select the shape of the capsid as

spherical, faceted, elongated and decapsidated, in the range of

parameters (directly related to measurable biophysical

parameters: bending constant and spontaneous curvature )

where a structure is stable.

[1] Howatson A.F. and Almeida J.D. 1960. Observations on the fine

structure of polyoma virus. Journal of Biophysical and Biochemical

Cytology, 8, 828-834.

[2] Kanesashi SN.et al 2003. Simian virus 40 VP1 capsid protein

forms polymorphic assemblies in vitro. Journal of General Virology,

84, 1899–1905.

[3] M.Aznar and D. Reguera. Physical ingredients controlling the

polymorphism and stability of viral capsid. In preparation.

50

P2. Optimizing a Combined Optical Tweezers-

Patch Clamp Set Up to Study ϕ-29 Connector

Rebeca Bocanegraa, Lara H. Moleiro

b, Francisco

Monroyb, José L. Carrascosa

a,c

aCentro Nacional de Biotecnología, CSIC, c/Darwin 3, Cantoblanco,

28049 Madrid, Spain b Departamento de Química Física I, Universidad Complutense,

28040 Madrid, Spain cInstituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA

Nanociencia), Cantoblanco, 28049 Madrid, Spain

Bacteriophage ϕ-29 encapsidates its DNA in a preformed

prehead using its packaging motor, located in one unique

vertex of the prehead. This packaging motor consists of three

macromolecular components: the connector protein, pRNA (an

RNA pentamer with structural function) and terminase (the

ATPase wich provides the energy for packaging from ATP

hydrolysis).

We have developed an optimized reconstitution

method for efficient orthogonal integration of native viral

connector into lipid bilayers, particularly of giant unilamelar

vesicles (1). We have optimized the bilayer in order to afford

the assembly of the complete ϕ-29 motor and we are currently

optimizing the DNA packaging in liposomes with integrated

connectors.

We also propose a new experimental set up based in

the combination of two powerful techniques: patch clamp and

optical tweezers. With this set-up we will be able to study the

forces implied in the DNA translocation through the channel,

by isolating a membrane patch with inserted connectors from

the GUVs previously formed.

51

1. L. H. Moleiro, I. López-Montero, I. Márquez, S. Moreno,

M. Vélez, J. L. Carrascosa and F. Monroy. ACS Synth. Biol 1(9),

414-424 (2012).

P3. Mechanical Disassembly of Single Virus

Particles Reveals Kinetic

Intermediates Predicted by Theory

Castellanos, M.a, Carrillo, P.J.P.

a, Pérez, R.

a, de Pablo

P.J. b, Mateu, M.G.

a

aCentro de Biología Molecular Severo Ochoa (Consejo Superior de

Investigaciones Científicas-Universidad Autónoma de Madrid) and bDepartamento de Física de la Materia Condensada C-III,

Universidad Autónoma de Madrid, Madrid, Spain.

New experimental approaches are required to detect the

conformational dynamics of viruses [1, 2] and elusive transient

intermediates predicted by simulations of virus assembly or

disassembly. We have used an atomic force microscope (AFM)

to mechanically induce partial disassembly of single

icosahedral T = 1 capsids and virions of the minute virus of

mice (MVM) [3]. The kinetic intermediates formed were

imaged by AFM. The results revealed that induced disassembly

of single MVM particles is frequently initiated by loss of one

of the 20 equivalent capsomers (trimers of capsid protein

subunits) leading to a stable, nearly complete particle that does

not readily lose further capsomers. With lower frequency, a

fairly stable, three-fourths-complete capsid lacking one

pentamer of capsomers and a free, stable pentamer were

52

obtained. The intermediates most frequently identified (capsids

missing one capsomer, capsids missing one pentamer of

capsomers, and free pentamers of capsomers) had been

predicted in theoretical studies of reversible capsid assembly

based on thermodynamic-kinetic models [4], molecular

dynamics [5], or oligomerization energies [6,7]. We conclude

that mechanical manipulation and imaging of simple virus

particles by AFM can be used to experimentally identify

transient, kinetic intermediates predicted by simulations of

assembly or disassembly.

1. M. Castellanos et al., PNAS 109, 12028-33 (2012).

2. M. G. Mateu, Virus Res. 168, 1-22 (2012).

3. M. Castellanos et al., Biophys J. 102, 2615-24 (2012).

4. S. Singh and A. Zlotnick, J. Biol. Chem. 278, 18249–55 (2003).

5. D. C. Rapaport, Phys. Rev. Lett. 101, 186101-4 (2008).

6. V. S. Reddy and Johnson, Adv. Virus Res. 64, 45–68 (2005).

7. V. S. Reddy et al., Biophys J. 74, 546–558 (1998).

53

P4. Comparative Study of Cellular

Modifications Induces by Adenovirus: Wild

Type, Packaging and Maturation Mutants

Gabriela Condezoa, Marta del Alamo

a, S. Jane Flint

b,

Miguel Chillónc, Carmen San Martín

a

aCentro Nacional de Biotecnología CNB-CSIC. Madrid (Spain)

bPrinceton University, Princeton, New Jersey (USA)

cCentro de Biotecnología Animal y Terapia Génica CBATEG-UAB.

Barcelona (Spain)

The maximum viral titer of human adenovirus type 5 (Ad5) is

obtained at 36hpi (hours post-infection). At this time of

infection, Ad5 has induced several well-characterized cellular

modifications. Ad5/FC31, an Ad5 mutant with two insertions

(attB/attP-ΦC31) flanking the packaging domain, has a

delayed viral cycle, 20 hours longer than wt (wild type);

however, its replication and protein expression is normal.

Studies showed that the delay is mainly affecting packaging of

the viral genome. We are taking advantage of this alteration in

the viral cycle to study adenovirus assembly within the cell.

Using electron microscopy (EM), we have compared changes

in the nuclear structure of cells infected with wt Ad5 or

Ad5/FC31. Apart from the changes previously described in the

bibliography, we observed a new structure specific for

Ad5/FC31 that we called “speckled bodies” (SBs) due to their

aspect at the electron microscope. SBs seem to contain subviral

particles trapped in DNA-rich regions, and their size varies in

range between 0.5 and 3 µm. Interestingly, SBs also appear in

cells infected with ts1, a mutant defective not in packaging but

in maturation. This observation suggests that packaging and

54

maturation could be coupled during adenovirus assembly. To

determine the composition of SBs, we have followed viral

DNA and DNA-packaging proteins in immune-fluorescence

assays. We are currently expanding the fluorescence study to

EM.

P5. Procapsids of Infectious Pancreatic

Necrosis Virus

Ana R. Correia

a, Daniel Luque

a, Natalia Ballesteros

b,

Sylvia R. Saint-Jeanb, Sara Pérez Prieto

b, JL Carrascosa

a

and JR Castóna

aCentro Nacional de Biotecnologia/CSIC, Department of Structures

of Macromolecules, Campus de Cantoblanco, c/ Darwin 328049

Madrid, Spain bICentro de Investigaciones Biologícas/CSIC, Department of

Molecular Microbiology, Ramiro de Maeztu 9, 28040 Madrid, Spain

Birnaviruses are nonenveloped dsRNA viruses with an

icosahedral T=13l capsid built of a single protein, VP2. Most

of our understanding of birnaviruses is based on studies of

infectious bursal disease virus (IBDV) and of infectious

pancreatic necrosis virus (IPNV). VP2 polymorphism is

controlled by an inherent switch, a transient C-terminal a-helix

in the precursor pVP21,2

. This switch is processed by viral and

cellular proteases3; cleavage takes place in a in a procapsid-like

structure stabilized by many copies of the VP3 scaffold

protein4,5. IPNV is a model for studying the coordination of

55

molecular factors involved in this multistep process. IPNV

procapsids, termed A particles, can be purified from IPNV-

infected BF-2 cells after 72 h in TNE buffer (10 mM Tris, 200

mM NaCl, 1 mM EDTA, pH 7.4). A mixture of immature and

mature particles (A and B, respectively) is purified if PES

buffer (25 mM PIPES, 150 mM NaCl, 20 mM CaCl2, p H 6.2)

is used throughout the purification process. A particles have

lower mobility than B particles in native agarose gels, although

they show the same protein composition. Both particle types

have a similar appearance by cryo-EM. B particles purified

from BF-2 cells have a similar electrophoretic mobility in

agarose gels to virions purified from CHSE cells using PES or

TNE buffers. Furthermore, A particles can be converted into B

particles after dialysis in PES buffer. Data suggest that

differences in pH and/or Ca2+

concentration are involved in

conformational changes in the capsid and might be associated

with distinct infectivities. This maturation mechanism, together

with other shared features, is reminiscent of the maturation

process triggered by acidic pH of nodavirus. 3D cryo-EM

analysis with A and B particles and IPNV virions are in

progress.

1. Saugar et al, Structure, 13, 1007 (2005)

2. Luque D. et al, J. Virol., 81 (13), 6869 (2007).

3. Irigoyen et al, JBC, 287(27), 24773 (2012)

4. Irigoyen et al, JBC, 284 (12), 8064 (2009)

5. Saugar et al, J. Biol. Chem. 285 (6), 3643 (2010);

56

P6. Structural Characterization Of The T7 Tail

Complex

Cuervo A., Pulido M., Martín-Benito J., Chagoyen M.,

Arranz R., Castón J.R., González-García V., García-Doval

C., Valpuesta J.M., van Raaij MJ. and Carrascosa J.L.

Department of Macromolecular Structure. Centro Nacional de

Biotecnología, CSIC. Darwin 3, Cantoblanco, 28049 Madrid, Spain.

Most of bacterial viruses need an specialised machinery named

the tail to deliver its genome inside the bacterial cytoplasm

without disrupting cellular integrity. T7 bacteriophage is a

well- characterized member of the Podoviridae bacteriophage

family infecting E. coli, and it presents a short non-contractile

tail that assembles sequentially in the viral head after DNA

packaging. T7 tail is a complex of around 2.5 MDa composed

by at least four proteins: connector (gp8), fibres (gp17) and the

tail tubular proteins (gp11 and gp12). Using cryo-electron

microscopy (Cryo-EM) and single particle image

reconstruction techniques we have determined the precise

topology of the tail structural proteins by comparing the

structure of the T7 tail extracted from viruses and a

recombinant complex formed by gp8, gp11 and gp12 proteins.

Furthermore, cloning and purification of the different tail

proteins allowed performing interaction assays to define the

location and the order of assembly of the proteins within the

complex. The existence of common folds among similar tail

proteins allowed to obtain pseudo-atomic threaded models of

the gp8 (connector) and gp11 (tubular) proteins, which were

mapped into the corresponding cryo-EM volumes of the tail

complex, generating a high resolution model of the connector-

57

gatekeeper interaction, and revealing the existence of a

common architecture with other Podoviridae tail complexes.

P7. Structural and functional characterization

of a non-cannonical replicase of ssRNA virus

(Thosea asigna virus): understanding

regulatory elements

Ferrero Dsa, Buxaderas M

a, Rodriguez JF

b, Verdaguer N

a

a Instituto de Biología Molecular de Barcelona-CSIC, c/Baldiri

Reixac 10, 08028, Barcelona . B

Centro Nacional de Biotecnología, c/Darwin 3, 28049, Madrid.

During infection most viruses employ a viral polymerase to

replicate and transcribe the viral genome. Do to their crucial

role, polymerases are broadly conserved in viruses following

the right hand architecture, with fingers, palm and thumb

subdomains. They also conserved six ordered sequence motifs

(A-B-C-D-E-F), four located into the palm subdomain (A to D)

and two (E-F) only present in RNA-dependent RNA

polymerases (RdRps). However a small group of ssRNA

viruses (Permutotetraviridae family) and some dsRNA viruses

(members of Birnaviridae family) not follow the canonical

organization, having a RdRp with a permuted palm motifs

organization (C-A-B-D). Given the shortage of this atypical

polymerases, there is scarce structural and biochemical

information about them.

Thosea asigna virus (TaV) is an insect restricted (+)ssRNA

58

virus that belong to the Permutotetravirus genus within the

Permutetetraviridae family. The paricular TaV RdRp, have a

non-canonical connectivity yielding a permuted palm

organization. In this work, we resolve the structure of TaV

RdRp and performed a biochemical characterization in order to

better understand this replicases.

The exhaustive analysis of the RdRp structure allow us to

identify several structural elements that potentially regulate the

polymerase activity. The amino terminus (30 aa) and an

extensive loop blocking the active site cavity may inhibit it.

They may undergo in a structural rearrengement, allowing the

polimerase to be active as we confirm by biochemical analysis.

The mutagenical analysis could gain insight into how RdRp

generally work and are regulated by their own structural

elements. In addition, we provide structural information to

support the existence of a common ancestor between ssRNA

and dsRNA viruses.

59

P8. The cryoEM reconstruction of Drosophila

C Virus (DCV) at 5.4 Å

Leandro Estrozi

a,, Jon Agirre

b, Jean-Luc Imler

c, Estelle

Santiagoc, Jorge Navaza

a, Guy Schoehn

a, Diego M.A.

u rinb

a Institut de Biologie Structurale Jean-Pierre Ebel. 41, rue Jules Ho-

rowitz F-38027 Grenoble Cedex 1, France. B Unidad de Biof sica and Fundaci n Biof sica Bizkaia. PO Box 644,

E-48080, Bilbao, Spain. C Institut de Biologie Molculaire et Cellulaire. 15 rue Rene Des-

cartes, F-67084 Strasbourg Cedex, France.

The Dicistroviridae family, which is currently classified under

the Picornavirales order, groups a pool of arthropod-infecting

viruses with bicistronic genomes. The interest in this family of

viruses has been fueled due to the economical implications of

their hosts, which range from beneficial arthropods (bees and

shrimps) to insect pests (crickets, ants and triatomines). Two

crystallographic structures of dicistroviruses have been report-

ed to date: Cricket Paralysis Virus (CrPV, type species of the

Cripavirus genus) and Triatoma Virus (TrV). Their structures

revealed that dicistroviruses share a core archetypal organiza-

tion, which is complemented by external and internal capsid-

wide differences that likely have arisen from unique host adap-

tation. In this work we report the cryoEM reconstruction at 5.4

Å resolution, and C-alpha trace of Drosophila C Virus (DCV),

a viral pathogen that infects Drosophila melanogaster, among

other Drosophila species. This virus holds a 65.8% sequence

identity with CrPV and, given the ability of the latter to repli-

60

cate in Drosophila hosts, a detailed comparison can give in-

sight into the infective cycle of dicistroviruses.

Keywords: cryoEM, reconstruction, dicistroviridae, DCV

P9. Mapping in vitro physical properties of

intact and disrupted virions at high resolution

using multi-harmonic atomic force microscopy

Mercedes Hernando-Pérez1 Alexander Cartagena

2, José

L. Carrascosa3, Pedro J. de Pablo

1, and Arvind Raman

2

1Departamento de Física de la Materia Condensada, Universidad

Autónoma de Madrid,

Madrid, Spain 2School of Mechanical Engineering and the Birck Nanotechnology

Center, Purdue University,

West Lafayette, IN, USA 3Centro Nacional de Biotecnología. CSIC, 28049 Madrid, Spain

Viruses are striking examples of macromolecular nano-

machines which carry out complex functions with minimalistic

structure. Understanding the relationships between viral mate-

rial properties (stiffness, charge density, adhesion, viscosity),

structure (protein sub-units, genome, receptors, appendages),

and functions (self-assembly, stability, disassembly, infection)

61

is of significant importance in physical virology and nanomed-

icine application (1-2).

We present quantitative maps at nanometer resolution of local

electro-mechanical force gradient, adhesion, and hydration

layer viscosity within individual Bacertiophage ɸ29 using the

multi-harmonic atomic force microscopy technique under

physiological condition. The technique significantly generaliz-

es recent multi-harmonic theory and enables high-resolution in

vitro quantitative mapping of multiple material (3).

High-resolution quantitative maps of bacteriophage ɸ29 show

that the material properties changes over the entire virion

provoked by the local disruption of its shell, providing

evidence of bacteriophage despressurization (4).

(1) Carrasco C, et al Proc. Natl. Acad. Sci. U. S. A. , (2006),

103:13706-13711

(2) T. Douglas and M. Young, Nature, 1998, 393, 152-155

(3) Raman A, et al. (2011) Nature Nanotech 6: 809-814

(4). Hernando-Pérez, M et al., Small, 2012, 8, 2365

62

P10. Evolutionary Dynamics of Genome

Segmentation in Multipartite Viruses

Jaime Iranzo and Susanna C. Manrubia

Centro de Astrobiología, INTA-CSIC, Ctra. de Ajalvir km. 4, 28850

Torrejón de Ardoz, Madrid, Spain

The origin and evolutionary history of viral genomes is a

classical problem that has inspired a long series of questions

and hypotheses in evolutionary biology. An especially

intriguing case concerns multipartite viruses, which are formed

by a variable number of genomic fragments packed in

independent viral capsids. This fact poses stringent conditions

on their transmission mode, demanding in particular a high

multiplicity of infection (MOI) for successful propagation.

Because the actual advantages of the multipartite viral strategy

are as yet unclear, the origin of multipartite viruses represents

an evolutionary puzzle. While classical theories suggested that

a faster replication rate or higher replication fidelity would

favour shorter segments, recent experimental results seem to

point to an increased stability of virions with incomplete

genomes as a factor able to compensate for the disadvantage of

mandatory complementation [1]. Using as main parameters

differential stability as a function of genome length and MOI,

we calculate the conditions under which a set of

complementary segments of a viral genome would outcompete

the non-segmented variant. Further, we examine the likeliness

that multipartite viral forms could be the evolutionary outcome

of the competition among the defective genomes of different

lengths that spontaneously arise under replication of a

63

complete, wild type genome [2]. We conclude that only

multipartite viruses with a small number of segments could be

produced in our scenario, and discuss alternative hypotheses

for the origin of multipartite viruses with more than four

segments [3].

1. S. Ojosnegros, J. García-Arriaza, C. Escarmís, S. C. Manrubia, C.

Perales, A. Arias, M. García Mateu and E. Domingo, PLoS Genet. 7,

e1001344 (2011).

2. J. García-Arriaza, S. C. Manrubia, M. Toja, E. Domingo and C.

Escarmís, J. Virol. 78, 11678-11685 (2004).

3. J. Iranzo and S. C. Manrubia, Proc. R. Soc. Lond. B. 279, 3812-

3819 (2012).

64

P11. Mechanical stability and reversible failure

of vault particles

A. Llauró1, P. Guerra

2, N. Irigoyen

3, J. F. Rodrí-

guez4, N. Verdaguer

2, P. J. de Pablo

1

1 Departamento de Física de la Materia Condensada, UAM,

Francisco Tomás y Valiente 7,28049-Madrid, Spain. 2Institut de Biologia Molecular de Barcelona, CSIC. Baldiri i Reixac

10, 08028-Barcelona, Spain. 3Division of Virology, Department of Pathology, University of

Cambridge, Tennis Court, Cambridge CB2 1QP, United Kingdom. 4Centro Nacional de Biotecnología, CSIC, Calle Darwin nº 3, 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, thus

removing any mark of the previous rupture. This

unprecedented systematic self-healing mechanism, which may

enable these particles to reversibly adapt to certain geometric

65

constraints, might help vaults safely pass through the nuclear

pore complex.

P12. Imaging and stiffness measurement of

IBDV virions by jumping mode AFM

Johann Mertensa, Santiago Casado

a, Carlos P. Mata

b,

Mercedes Hernando-Perézc, Pedro J. De Pablo

c, José R.

Castónb and José L. Carrascosa

a,b

aIMDEA Nanociencia, Unidad asociada CNB-IMDEA

Nanociencia, Campus de Cantoblanco, 28049 Madrid, Spain. bCentro Nacional de Biotecnología - CSIC. Darwin 3,

Campus de Cantoblanco, 28049 Madrid, Spain. cDpto. de Física de la Materia Condensada, Universidad

Autonoma de Madrid, Campus de Cantoblanco, 28049

Madrid, Spain.

We imaged surface-attached IBDV virions by using jumping-

mode AFM, which allowed us to control maximal tip-sample

forces accurately. Six natural populations of the virus (E1 to

E6), which share a similar protein composition but increasing

copy number of genome segments inside the viral capsid, have

been identified and probed separately. Our results show that

one can probe nanoscale IBDV shells and quantitatively extract

their mechanical properties. This constitutes the first direct

evaluation of the mechanical properties of IBDV capsids.

Surprisingly, the stiffness of the capsids changes and seems to

increase with the amount of RNA packed inside the virus, from

66

0.4 N/m for E1 to 0.78 N/m for E6. Mechanical reinforcement

of IBDV capsids is a new feature to be explored in relation

with their biochemical properties and almost to understand the

variations in the infectivity of the virus.

P13. Monitoring Dynamics of Human

Adenovirus Disassembly Induced by

Mechanical Fatigue

A. Ortega-Esteban

a, A. J. Pérez-Berná

b, R. Menéndez-

Conejerob, S. J. Flint

c, C. San Martín

b and P. J. de Pablo

a

aDepartamento de Física de la Materia Condensada, Universidad

Autónoma de Madrid, 28049 Madrid, Spain bDepartment of Macromolecular Structure, Centro Nacional de

Biotecnología (CNB-CSIC). Darwin 3, 28049 Madrid, Spain cDepartment of Molecular Biology, Princeton University, Princeton,

NJ 08544, USA

The standard pathway for virus infection of eukaryotic cells

requires disassembly of the viral shell to facilitate release of

the viral genome into the host cell. Here we use mechanical

fatigue, well below rupture strength, to induce stepwise

disruption of individual human adenovirus particles under

physiological conditions, and simultaneously monitor

disassembly in real time. Our data show the sequence of

dismantling events in individual mature (infectious) and

immature (noninfectious) virions, starting with consecutive

release of vertex structures followed by capsid cracking and

67

core exposure. Further, our experiments demonstrate that

vertex resilience depends inextricably on maturation, and

establish the relevance of penton vacancies as seeding loci for

virus shell disruption. The mechanical fatigue disruption route

recapitulates the adenovirus disassembly pathway in vivo, as

well as the stability differences between mature and immature

virions.

2. A. Ortega-Esteban, A. J. Pérez-Berná, R. Menéndez-Conejero, S. J.

Flint, C. San Martín and P. J. de Pablo, Sci. Rep. 3, 1434 (2013).

P14. The Non-icosahedral Components in

Adenovirus Studying By Cryo-electron

Tomography

Pérez-Berná AJ

a, Chichón FJ

a,Fernández JJ

a, Winkler D

b,

FontanaJb, Flint SJ

c, Carrascosa JL

a, Steven AC

b, San

Martín Ca

aCentro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain

bIAMS, National Institutes of Health, Bethesda, Maryland,

cPrinceton University, Princeton, New Jersey

Adenovirus has a non-enveloped icosahedral capsid enclosing

a 35 kbp linear dsDNA genome associated with ~25 MDa of

DNA-binding proteins, making up a non-icosahedral core. We

are using cryo-electron tomography to visualize the non-

icosahedral elements of adenovirus. We have extracted, aligned

and classified the vertex regions from 612 individual virus

68

tomograms using maximum-likelihood subtomogram

averaging methods. This procedure revealed that the vertices in

each icosahedral virion can be categorized in three groups,

according to the relation between the shell and the internal

contents. In each viral particle, one vertex is in direct contact

with the core, while the opposed vertex presents a gap between

the icosahedral shell and the core, and the other 10 vertices

present an intermediate situation. This observation may

indicate the presence of additional proteins beneath one

singular vertex (eg the packaging machinery), or an asymmetry

in the distribution of the genome and accompanying proteins

within the virion. Additionally, our cryo-electron tomography

analysis of adenovirus shows that each particle contains 150-

180 discrete ellipsoidal densities with asymmetrical

distribution profile with approximate radii 14 x 6 nm. This is

the first time that the “adenosomes” have been directly

observed within the virion.

69

P15. Electrostatic repulsions at neutral pH

underlie the weak thermal stability of foot-and-

mouth disease virus, and guide the engineering

of modified virions of increased stability for

improved vaccines

Rincón Va, Rodríguez-Huete A

a, Harmsen MM

b and

Mateu MG

a

aCentro de Biología Molecular “Severo Ochoa” (CSIC-UAM),

Universidad Autónoma de Madrid, Cantoblanco, 28049. Madrid,

Spain bCentral Veterinary Institute of Wageningen UR, P.O. Box 65, 8200

AB Lelystad, The Netherlands

We are investigating the molecular basis of physical stability of

virus particles in order to understand virus assembly, stability

and dynamics, and also for bio-nanotechnological purposes

including thermostable vaccines. One of our model systems is

foot-and-mouth disease virus (FMDV), the causative agent of

one of the economically most important animal diseases

worldwide. In the present study we have investigated the

molecular mechanism by which mutation A2065H in capsid

protein VP2 exerts a greatly thermostabilizing effect on the

virion against dissociation into pentameric subunits. The

results have revealed the presence in the virion of coulombic

repulsions between pentamers, even at neutral pH, which

contribute to explain the low thermostability of FMDV and its

empty capsid. Several acidic residues not far from residue

A2065 contribute to this repulsion. Most likely, mutation

A2065H stabilizes the virion because the additional positive

70

charge introduced may partly neutralize some of the excess

negative charge around, thus weakening the interpentameric

repulsion. The discovery of this repulsive effect between

pentamers at neutral pH allowed us to undertake a new rational

protein engineering approach on FMDV that led to obtain four

virus variants of increased thermostability. These engineered

FMDVs constitute good candidates for development of

thermostable vaccines against FMD based on virions or empty

capsids.

71

P16. An evolutionary systemic approach to

virus-host interactions

Guillermo Rodrigoa, Javier Carrera

a, and Santiago F.

Elenaa

aInstituto de Biología Molecular y Celular de Plantas, Consejo

Superior de Investigaciones Científicas - Universidad Politécnica de

Valencia, València, Spain.

Understanding the mechanisms by which plants trigger host

defenses in response to viruses has been a challenging problem

owing to the multiplicity of factors and complexity of

interactions involved. The advent of genomic techniques,

however, has opened the possibility to grasp a global picture of

the interaction. Here, we used Arabidopsis thaliana to identify

and compare genes that are differentially regulated upon

infection with seven distinct (+)ssRNA and one ssDNA plant

viruses. In a first approach, we established lists of genes

differentially affected by each virus and compared their

involvement in biological functions and metabolic processes.

We found that phylogenetically-related viruses significantly

alter the expression of similar genes and that viruses naturally

infecting Brassicaceae display a greater overlap in the plant

response. In a second approach, virus-regulated genes were

contextualized using models of transcriptional and protein-

protein interaction networks of A. thaliana. Our results confirm

that host cells undergo significant reprogramming of their

transcriptome during infection, which is possibly a central

requirement for the mounting of host defenses. We uncovered a

general mode of action in which perturbations preferentially

affect genes that are highly connected, central and organized in

72

modules.

73

LIST OF PARTICIPANTS

Last Name First Name Email

Abian Olga oabifra@unizar.es

Abrescia Nicola G nabrescia@cicbiogune.es

Arribas Hernán María arribashm@inta.es

Aznar Palenzuela María maznar@ffn.ub.es

Bittner Alexander a.bittner@nanogune.eu

Blanc Stéphane blanc@supagro.inra.fr

Bocanegra Rojo Rebeca rbocanegra@cnb.csic.es

Briones Carlos cbriones@cab.inta-csic.es

Cabanillas Laura lcabanillas@cab.inta-csic.es

Carrascosa Jose L. jlcarras@cnb.csic.es

Casado Santiago santiago.casado@imdea.org

Castrillo Briceño Mariana mariana.castrillo@cnb.csic.es

Catalan Pablo pcatalan@math.uc3m.es

Condezo Castro Gabriela gncondezo@cnb.csic.es

Cordoba Garcia Laura lcordoba@cnb.csic.es

Cuervo Gaspar Ana acuervo@cnb.csic.es

Cuesta Jose cuesta@math.uc3m.es

de la Escosura Navazo

Andrés andres.delaescosura@uam.es

de Pablo Gómez Pedro José p.j.depablo@uam.es

Domingo Esteban edomingo@cbm.uam.es

Elena Santiago sfelena@ibmcp.upv.es

Fernández Arias Clemente clmntf@yahoo.com

Ferrero Diego Sebas-tián

dferrero@cnb.csic.es

Fuertes Miguel Angel mafuertes@cbm.uam.es

Garcia Doval Carmela carmela.garcia@cnb.csic.es

García-Mateu Mauricio mgarcia@cbm.uam.es

Granell Meritxell mgranell@cnb.csic.es

Guérin Diego M.A. diego.guerin@ehu.es

74

Hernández Rojas Javier jhrojas@ull.es

Hernando Mercedes mercedes.hernando@uam.es

Iranzo Sanz Jaime iranzosj@cab.inta-csic.es

Lázaro Ester lazarole@cab.inta-csic.es

Llauró Portell Aida aidallauro@gmail.com

Luque Buzo Daniel dluque@isciii.es

Manrubia Susanna scmanrubia@cab.inta-csic.es

Mertens Johann jmertens@imm.cnm.csic.es

Nguyen Thanh th.nguyen@cnb.csic.es

Ortega Esteban Alvaro alvaro.ortega@uam.es

P. Mata Carlos cperez@cnb.csic.es

Pallàs Benet Vicente vpallas@ibmcp.upv.es

Perales Viejo Celia cperales@cbm.uam.es

Perez Berna Ana Joaquina ajperezberna@gmail.com

Pérez Carrillo Pablo José pjperez@cbm.uam.es

Ramasco Jose J. jramasco@ifisc.uib-csic.es

Reguera David dreguera@ub.edu

Rey Felix rey@pasteur.fr

Rincón Forero Verónica Del Pilar

vrincon@cbm.uam.es

Rodrigo Guillermo guirodta@gmail.com

Rodriguez Alicia alicia_r@cbm.uam.es

Rodríguez Dolores drodrig@cnb.csic.es

Rodriguez Martinez Javier Maria j.rodriguez@isciii.es

Rubí J. Miguel mglrb2@gmail.com

Ruiz Castón José jrcaston@cnb.csic.es

Ruiz Herrero Teresa teresa.ruiz@uam.es

San Martín Carmen carmen@cnb.csic.es

Sanz Gaitero Marta martasanz34@gmail.com

Siber Antonio asiber@ifs.hr

Singh Abhimanyu abhimanyu.singh@cnb.csic.es

Valbuena Jiménez Alejandro avalbuena@cbm.uam.es

van Raaij Mark mjvanraaij@cnb.csic.es

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Vega Sonia svega@bifi.es

Velazquez-Campoy

Adrian adrianvc@unizar.es

Verdaguer Nuria nvmcri@ibmb.csic.es

Viegas Correia Ana Raquel arviegas@cnb.csic.es

Organizer:

P. J. de Pablo Gómez p.j.depablo@uam.es

Departamento de Física de la Materia Condensada

Facultad de Ciencias

Universidad Autónoma de Madrid

Coordinator:

David Reguera dreguera@ub.edu

Departament Física

Fonamental

Universitat de

Barcelona

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