1. Studies on the Role of Four Cysteines of SV40 in Infectious Virion Formation Wafa Mana Dr. E. Gharakhanian California State University, Long Beach

2. Outline Background Goals of research Materials and Methods Results Conclusion Future experiments Acknowledgments

3. 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

4. (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

5. (SV40) cont… Role of cysteines 49, 87, 254, and 267 in virion viability investigated by generating single-point cysteine-to-serine mutants (to disrupt disulfide bonds), and transfecting mutant DNA into CV-1 cells Role of cysteines 49, 87, 254, and 267 in virion viability investigated by generating single-point cysteine-to-serine mutants (to disrupt disulfide bonds), and transfecting mutant DNA into CV-1 cells Mutants resulting in viable virions show cytopathogenic effect and plaque formation. Mutants resulting in viable virions show cytopathogenic effect and plaque formation.

6. 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

7. 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

8. 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

9. M&M cont… Serial dilution infections 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

10. Results Normal progression of SV40 infection as seen in CV-1 cells NormalCPE Plaques

11. Results SV40 DNACPE?Plaques? SV40-wtYes SV40-C49S (2)Yes SV40-C87S (3)Yes SV40-C254S (6)No SV40-C267S (7)Yes SV40-C49S, C87S (2,3)No SV40-C254S-C267S (6,7)Yes SV40-C49S,C87S,C254S, C267S (4X) No SV40-mockNo Plaque assay: CPE and plaques indicate virus is viable

12. Results SV40-wtSV40-2SV40-3SV40-2,3 SV40-6SV40-7SV40-6,7SV40-4X SV40- MOCK Plaque assay: areas of cleared cells are plaques.

13. Results Plaque counts for wt & mutants showing plaques SV40Mean plaque sizeSE meanTitres WT2.7500.2928.0 x 10 3 22.9580.3157.0 x 10 3 32.0180.3806.4 x 10 3 72.5580.2877.2 x 10 3 6,72.5670.2907.2 x 10 3 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.

14. 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

15. 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 H 0 that there is no significant variation btwn average plaque sizes. Titres: one-way ANOVA p >  =0.05 so no significant variation btwn titres.

16. 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

17. Acknowledgments Dr. Editte Gharakhanian Manith Norng The Howard Hughes Medical Institute

18. 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

19. 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.

20. 9) Nakanishi, A., Clever, J., Li, P., Kasamatsu, H., Yamada, M. 1996. Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA. PNAS 93:96-100 9) Nakanishi, A., Clever, J., Li, P., Kasamatsu, H., Yamada, M. 1996. Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA. PNAS 93:96-100 10) Salunke, D. M., Caspar, D. L. D., Garcea, R. L. 1986. Self- assembly of purified polyomavirus capsid protein VP1. Cell. 46:895-904. 10) Salunke, D. M., Caspar, D. L. D., Garcea, R. L. 1986. Self- assembly of purified polyomavirus capsid protein VP1. Cell. 46:895-904. 11) Tooze, J. 1980. DNA tumor viruses. Cold Spring Harbor Laboratory. 2d ed. 11) Tooze, J. 1980. DNA tumor viruses. Cold Spring Harbor Laboratory. 2d ed. http://www.photo-guide.com/science.html http://www.photo-guide.com/science.html http://www.photo-guide.com/science.html http://creative.gettyimages.com/source/search/resultsmain.asp?t xtSearch=Science&source=quickSearch&brand=pdi http://creative.gettyimages.com/source/search/resultsmain.asp?t xtSearch=Science&source=quickSearch&brand=pdi http://creative.gettyimages.com/source/search/resultsmain.asp?t xtSearch=Science&source=quickSearch&brand=pdi http://creative.gettyimages.com/source/search/resultsmain.asp?t xtSearch=Science&source=quickSearch&brand=pdi