Role of Simian Virus 40 Vp1 Cysteines in Virion Infectivity

Abstract

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structurebased analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-typetransfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca 2؉ -binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca 2؉ ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

Importance of Vp1 Calcium-Binding Residues in Assembly, Cell Entry, and Nuclear Entry of Simian Virus 40

“…We have previously shown that mutating Cys254, a residue located on a conserved loop near the calcium sites, can severely reduce the formation of infectious particles. That this defect is observed for substitution into certain side chains (leucine, glycine, serine, and arginine) but not into alanine is consistent with the local structure of Cys254, including the putative calcium-binding residues, being important for particle assembly and infectivity (17). Mutating certain acidic residues of the calcium-binding sites also affects the shape or stability of virus-like particles (VLPs) formed in Vp1-expressing insect cells (12).…”

“…Viability was determined by plaque formation assays using serial dilutions of lysates prepared by freezethawing NO-SV40 DNA-transfected CV-1 monkey kidney cells at 72 h posttransfection, as described previously (17,18). For select NO-SV40 mutants, plaques were lifted from the culture dish, resuspended in 0.1 to 1.0 ml of TD buffer, and lysed by freeze-thawing.…”

“…The extent of viral DNA replication was determined by gel quantitation of DpnI-resistant viral DNA extracted from 1 ϫ 10 6 transfected cells, and Vp1 production was assessed by anti-Vp1 Western blot analysis of 5 ϫ 10 4 transfected cells, as described previously (17,18).…”

SV40 reporter viruses

“…The presence of mature viral particles was detected by sedimentation velocity ultracentrifugation as previously reported (Li et al, 2000). Briefly, nuclei were isolated from infected cells, sonicated and overlayed onto 5-32% sucrose gradients (50 mM N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) buffer, pH 7.5) in a SW41 tube.…”

Roles of Cellular Redox Factors in Pathogen and Toxin Entry in the Endocytic Pathways

“…The pentamers are linked to each other by the interchain disulfide bonds between the residues Cys104. Isomerization of the disulfide bonds in the ER is crucial for the viral uncoating process [136][137][138]. Recent data has shown that SV40 makes use of the protein folding and quality control machinery in the ER for initial uncoating and membrane translocation [46].…”