Studies on the Role of Four Cysteines of SV40 in

Infectious Virion Formation

Wafa ManaDr. 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 10ulVirions 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

Normal progression of SV40 infection as seen in CV-1 cells

Normal CPE Plaques

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

Acknowledgments

Dr. Editte Gharakhanian Manith Norng The Howard Hughes Medical Institute

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