Characterization of the Archaeal Thermophile Sulfolobus

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