Importance of Calcium-Binding Site 2 in Simian Virus 40 Infection

Abstract

The exposure of molecular signals for simian virus 40 (SV40) cell entry and nuclear entry has been postulated to involve calcium coordination at two sites on the capsid made of Vp1. The role of calciumbinding site 2 in SV40 infection was examined by analyzing four single mutants of site 2, the Glu160Lys, Glu160Arg, Glu157Lys (E157K), and Glu157Arg mutants, and an E157K-E330K combination mutant. The last three mutants were nonviable. All mutants replicated viral DNA normally, and all except the last two produced particles containing all three capsid proteins and viral DNA. The defect of the site 1-site 2 E157K-E330K double mutant implies that at least one of the sites is required for particle assembly in vivo. The nonviable E157K particles, about 10% larger in diameter than the wild type, were able to enter cells but did not lead to T-antigen expression. Cell-internalized E157K DNA effectively coimmunoprecipitated with anti-Vp1 antibody, but little of the DNA did so with anti-Vp3 antibody, and none was detected in anti-importin immunoprecipitate. Yet, a substantial amount of Vp3 was present in anti-Vp1 immune complexes, suggesting that internalized E157K particles are ineffective at exposing Vp3. Our data show that E157K mutant infection is blocked at a stage prior to the interaction of the Vp3 nuclear localization signal with importins, consistent with a role for calcium-binding site 2 in postentry steps leading to the nuclear import of the infecting SV40.

ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation

“…The negative-stain EM projection images of the structure of SV40 revealed that the majority of the virus (Ͼ93%) appeared as near-spherical particles ϳ45 nm in diameter, as previously described (40,41) (Fig. 3B and C and 4).…”

Viral calciomics: Interplays between Ca2+ and virus

“…Interestingly, some of the mutants fail to enter the cell (e.g., E330K in Site 1) while others enter the cells, but fail to migrate to the nucleus (e.g., E157A-E160A and E216K in Site 2) due to an inability to bind Ca 2+ leading to premature dissociation. Thus, the binding of two Ca 2+ ions to the dual Ca 2+ -binding site on VP1 is essential for the assembly of the icosahedral capsid, as well as the cell entry and nuclear transport [103] , [119] .…”

Structure and Function of a Genetically Engineered Mimic of a Nonenveloped Virus Entry Intermediate

“…The removal of Ca 2ϩ from capsids could be particularly crucial for viruses that depend on metal ions for assembly and stability, such as rotavirus, mouse polyomavirus, and dragon grouper nervous necrosis virus (DGNNV) (1,39,43). A few studies have reported that mutations at the metal-binding sites decrease virus infectivity by impairing the early stages of virus-host interaction and genome release (23,24). When Ca 2ϩ is removed from the capsids of plant viruses such as cowpea chlorotic mottle virus (CCMV), tomato bushy stunt virus (TBSV), and turnip crinkle virus (TCV) by metal chelators (2,33,35), the virion swells by almost 10% and becomes susceptible to RNase.…”