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Studies on the Role of Four Cysteines of SV40 in
Infectious Virion Formation
Wafa Mana
Dr. E. Gharakhanian
California State University, Long Beach
Outline
Background Goals of research Materials and Methods Results Conclusion Future experiments Acknowledgments
Simian Virus 40 (SV40) DNA papovavirus of the genus
polyomavirus First discovered as contaminant of
African Green monkey kidney cells used for growing poliovirus for vaccine purposes
SV40 possible vector in gene therapy
Double-stranded 5.3 kb long genome
Three structural proteins VP1, VP2, VP3 compose the icosahedral capsid
(SV40) cont…
Main structural protein VP1 composes pentamers
Five arms emerge from each pentamer and insert into neighboring pentamers
Disulfide bonds between 7 cysteine residues in VP1 pentamers hold capsid together. 3 cysteines implicated in disulfide bond formation
(SV40) (SV40) cont…cont…
Role of cysteines 49, 87, 254, Role of cysteines 49, 87, 254, and 267 in virion viability and 267 in virion viability investigated by generating investigated by generating single-point cysteine-to-serine single-point cysteine-to-serine mutants (to disrupt disulfide mutants (to disrupt disulfide bonds), and transfecting mutant bonds), and transfecting mutant DNA into CV-1 cellsDNA into CV-1 cells
Mutants resulting in viable Mutants resulting in viable virions show cytopathogenic virions show cytopathogenic effect and plaque formation.effect and plaque formation.
Goals of Research
Confirm role of cysteines 49, 87, 254, and 267 in virion viability
Determine mean plaque sizes/ SV40 mutant Determine titre (p.f.u./plaque) / SV40 mutant
through the use of serial dilution transfections
Materials and methods
Culture SV40 mutants and wt: use E. coli vector
Purify pSV40 using extraction kit
Digest plasmid: use BamHI
Separate fragments: run DNA on 1% agarose gel. Remove 5.3 kb band (SV40).
Extract SV40 DNA from gel.Check conc @ 260 nm; need conc 0.1-1.0ug/ul
SV40 DNA purification
M&M cont…
Transfections
Add 3ug DNA ( w/lipid in DMEM sln) to each plate of CV-1 cells. Each mutant has a set of 3 plates
Day 14: stain cells w/crystal violet
Day 7: CPE? Day 10: plaques?
Plug of plaques. Harvest virions for infections
M&M cont…
Serial dilution infections
10ul 10ul 10ul
Virions harvested from 1 plug are diluted in DMEM to 1/10, 1/100, & 1/1000 dilutions
Dilutions added to CV-1 cells
Plaques/plate recorded
Results
SV40 DNA CPE? Plaques?
SV40-wt Yes Yes
SV40-C49S (2) Yes Yes
SV40-C87S (3) Yes Yes
SV40-C254S (6) No No
SV40-C267S (7) Yes Yes
SV40-C49S, C87S (2,3) No No
SV40-C254S-C267S (6,7) Yes Yes
SV40-C49S,C87S,C254S, C267S (4X)
No No
SV40-mock No No
Plaque assay: CPE and plaques indicate virus is viable
Results
SV40-wt SV40-2 SV40-3 SV40-2,3
SV40-6 SV40-7 SV40-6,7 SV40-4X SV40-MOCK
Plaque assay: areas of cleared cells are plaques.
ResultsPlaque counts for wt & mutants showing plaques
SV40 Mean plaque size SE mean Titres
WT 2.750 0.292 8.0 x 103
2 2.958 0.315 7.0 x 103
3 2.018 0.380 6.4 x 103
7 2.558 0.287 7.2 x 103
6,7 2.567 0.290 7.2 x 103
One-way ANOVA on mean plaque sizes: =0.05, p value=0.417. One-way ANOVA on mean titres: =0.05, p value=0.726.
Discussion
Plaque assays: confirmation of previous results: mutants w/ cys-ser changes at cysteines 49, 87, and 267 show CPE and plaque formation. These cysteines are non-essential for viable virion formation.
Cys 267 mutant showed no plaques: this cysteine is essential for disulfide bond formation.
No plaques formed in SV40-C49S, C87S implies that virus requires presence of at least one cysteine for formation of viable virions
Discussion cont…
Double mutant SV40-C254S, C267S showed plaque formation while SV40-C254S showed no plaques…?
Plaque counts: one-way ANOVA p > =0.05, so accept H0 that there is no significant variation btwn average plaque sizes.
Titres: one-way ANOVA p > =0.05 so no significant variation btwn titres.
Future Research
What is going on with double mutant SV40-C254S, C267S? How is a viable virus able to form with a double mutation when it can’t form with a single mutant? Do in vitro study in cell-free lysate to check for capsid formation.
Visualization of SV40 mutants’ protein structure esp. SV40-C267S and SV40-C254S, C267S by x-ray crystallography
References
1) Consigli, R. A., Cai, X., Chang, D., Gillock, E. T., Rottinghaus, S., Smiley, K. A. 1997. Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the Bacilovirus system.
2) Fasching C. L., Gharakhanian E., Orlando, S. J. 2001. Cys9, Cys104, and Cys207 of simian virus 40 vp1 are essential for infectious virion formation in CV-1 cells. J. Gen. Vir. 82:1935-1939.
3) Garcea. R. L., Caspar, D. L. D., Salunke, D. M. 1987. Site-directed mutation affecting polyomavirus capsid self-assembly in vitro. Nature. 329:86-87.
4) Handa, H., Han, S., Hoque, M., Ishizu, K., Kanesashi, S., Kataoka, K., Watanabe, H. Roles of disulfide linkage and calcium ion-mediated interactions in assembly and disassembly of virus-like particles composed of simian virus 40 VP1 capsid protein. J. Gen. Vir. 75:61-72
References
5) Jao, C. C., Gharakhanian, E., Perez, A. R, Weidman, M. K. 1999. Cys9, Cys104, and Cys207 of simian virus 40 VP1 are essential for inter-pentamer disulfide-linkage and stabilization in cell-free lysate. J. Gen. Vir. 80:2481-2489.
6) Kasamutsu, H., Liddington, R. C., Nakanishi, A., Peggy, L.P., Tran, M. A., Salazar, A. M. 2000. Role of simian virus 40 VP1 cysteines in virion infectibility. J. Vir. 74:11388-11393.
7) Liddington, R. C., Benjamin, T. L., Harrison, S. C., Moulai, J., Sahli, R., Yan, Y. 1991. Structure of simian virus 40 at 3.8-Å resolution. Nature 354:278-284.
8) Oppenheim, A., Sandalon, Z. 1997. Self-assembly and protein-protein interactions between the SV40 capsid proteins produced in insect cells. Virology. 237:414-421.
9) 9) Nakanishi, A.Nakanishi, A., , Clever, J., Li, P., Kasamatsu, H., Yamada, Clever, J., Li, P., Kasamatsu, H., Yamada, M.M. 1996. Association with capsid proteins promotes nuclear 1996. Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA. PNAS targeting of simian virus 40 DNA. PNAS 93:93:96-10096-100
10) 10) Salunke, D.Salunke, D. M., Caspar, D. L. D., Garcea, R. L.M., Caspar, D. L. D., Garcea, R. L. 1986. Self- 1986. Self-assembly of purified polyomavirus capsid protein VP1. Cell. assembly of purified polyomavirus capsid protein VP1. Cell. 46:46:895-904.895-904.
11) 11) Tooze, J.Tooze, J. 1980. DNA tumor viruses. Cold Spring Harbor 1980. DNA tumor viruses. Cold Spring Harbor Laboratory. 2d ed. Laboratory. 2d ed.
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