Characterization of the ArchaealThermophile Sulfolobus TurretedIcosahedral Virus Validates an
Evolutionary Link among Double-Stranded DNA Viruses from All
Domains of Life
Presenter: Justin BradshawCo-Presenter: Alice Mei Lee
Viruses_ Latin, poison
_ Non-living (?) obligateparasites
_ Possible Origins
_ Acytota: Possibledomain
_ Classified using ICTV,Baltimore
Orthopoxvirus Cowpox virus
Viral Phylogeny
_ Absent from fossil record
_ No rRNA
_ Topological similarity used to determinecommon ancestry
_ DI Stuart et. al. uses a PHYLIP analysis ofprobabilities of equivalence between pairs ofresidues for viral proteins responsible for thecapsid shell (pretty picture)
PRD1
_ dsDNA bacteriophage
_ Icosahedral
_ Lipid bilayer
_ Pentameric “spikes” aticosahedral facets
Sulfolobus solfataricus P2
_ Archaeum isolatedfrom many volcanicareas (~80°C, pH ~2-3)
_ Lithoautotrophic orchemoheterotrophic
_ tetraether lipidmonolayer
Sulfolobus Turreted Icosahedral Virus
_ 17,663 bp dsDNAgenome
_ 36 predicted ORFs
_ Infects S. SolfataricusP2
STIV as a PRD1-like virus
_ Almost identical proteinfold structure
_ STIV “turrets” similar toPRD1 “spikes”
_ Inner electron-denselayers similar to PRD1lipid layer
_ What say proteomics?
SDS-PAGE
Gygi and Aebersold Current opinion inchemical biology 2000
Schematic of a standard proteome analysis by 2DE-MS
•Virus purification
•SDS-PAGE/IEF +SDS-PAGE/In-gel digestion
•MALDI-TOF MS
•Nanospray LC-MS/MS•Homology searches for STIV structural proteins
•Protein modeling
•Glycoprotein analysis
•Lipid Analysis
Methods
MALDI-TOF MS/MS
ESI MSElectroSpray Ionization Mass spectrometry
• Produces gaseous ionized molecules from a liquidsolution
• Done by creating a fine spray of highly chargeddroplets in the presence of a strong electric field.
http://www.newobjective.com/electrospray/
Nano ESI (LC-MS/MS)
Siuzdak Mass spectrometry for biotechnology 1996
•Droplets <10_m in diameter
•Flowrate is 20-40nl/min
Nano ESI (LC-MS/MS)
Siuzdak Mass spectrometry forbiotechnology 1996
Nano ESI Needle point
www.sitemaker.umich.edu/.../files/esi_spray.jpg
Agilent XCT-plus ion trap massspectrometer
www.chemistry.gsu.edu/facilities/Agilent_LC-MS02.JPG
Tandem MS (MS/MS)
Siuzdak Mass spectrometry for biotechnology 1996
Siuzdak Mass spectrometry for biotechnology 1996
Structural prediction used to identifyproteins
Host Proteins
_ SS07D – Stabilizes and packages viral DNA?
− Suggested histone-like role in S. solfataricus
− Similar protein in SSV1
− Needed: Similar proteins in other virus particles
_ SSO0881 – Cell sorting and trafficking?
− Similar protein in yeast and mammals
_ Consistent cosedimentation, but can't rule outcontamination
Genome Mapping
_ A78 N-terminus peptideis a valine located 9nucleotides downstreamfrom A55 amber stopcodon (UAG)
_ Read-through product?
_ Alternative start codon?
PSI-BLAST
_ Position Specific Iterative BLAST (Basic LocalAlignment Search Tool)
_ Position specific scoring
_ Scoring based on conservation in relatedproteins
_ “related proteins” determined using traditionalBLAST results
_ Repeats iteratively
PSI-BLAST Results for B164
Surface Overlap Maximization
Consistently placed a single copy of B164 at base of turretcomplex
B164 as a nucleic acid-packagingATPase
Three lines of indirectevidence:
_ Profile-ProfileComparision
_ Position
_ 3-nm channel
C381, A223, C557
_ Structure and folding of C381, A223 similar to
PRD1's P5 vertex protein
_ High mass in 1D SDS-PAGE, expected mass in2D – indication of complex (C381 and A223)
_ C557 also located in turrets
_ Turret mass ~637 kDa
_ C557 + C381 + A223 ~125 kDa (pentameric?)
Glycosylation
_ Stained with Pro-Q
_ B345 Glycosylated
_ Deglycosidasesineffective
_ Too stable?
STIV contains lipids
Analysis of lipids
Lipids
_ Outer leaflet interacts with B345 (acidic lipids,basic C-terminal of B345)
_ Inner leaflet function unknown
− Thermal stability?
− Particle assembly/disassembly?
− Capsid assembly?
Direct Paper Conclusion
Protein, lipid andcarbohydrate analysis ofSTIV indicates that it is
similar to tail-less dsDNAviruses from the other
domains of life
Extended Paper Conclusions
_ Proteomics-based approaches to proteinidentification have much utility
_ Relationships between viral proteins can onlyreliably be detected structurally
_ Strengthens evidence for existence of acommon viral ancestor that predates theEukarya/Bacteria/Archaea divergence