Association of Simian Virus 40 Vp1 with 70-Kilodalton Heat Shock Proteins and Viral Tumor Antigens

Abstract

Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked intermediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be coimmunoprecipitated with Vp1. Studies in vitro demonstrated the ATP-dependent interaction of Vp1 and cellular chaperones. Interestingly, viral cochaperones LT and ST were essential for stable interaction of HSP70 with the core Vp1 pentamer Vp1 (22-303). LT and ST also coimmunoprecipitated with Vp1 in vivo. In addition to these identified (co)chaperones, stable, covalently modified forms of Vp1 were identified for a folding-defective double mutant, C49A-C87A, and may represent a "trapped" assembly intermediate. By a truncation of the carboxyl arm of Vp1 to prevent the Vp1 folding from proceeding beyond pentamers, we detected several apparently modified Vp1 species, some of which were absent in cells transfected with the folding-defective mutant DNA. These results suggest that transient covalent interactions with known or unknown cellular and viral proteins are important in the assembly process.Simian virus 40 (SV40), a polyomavirus, has an icosahedral capsid whose structure is known at atomic resolution (34, 48). The capsid is built from a major capsid protein (Vp1) and two minor, internally embedded capsid proteins (Vp2 and Vp3). One Vp1 monomer folds into a core ␤-barrel domain of jelly roll topology along with the N-and C-terminal arms. Five Vp1 monomers interdigitate their secondary structures to form pentamers that are tied together via interactions of their Cterminal arms (34). Little is known about how Vp1 folds into the icosahedral structure. Vp1 folds in the cytoplasm of infected host cells first through monomeric intermediates and then through oligomeric intermediates that contain transitory disulfide bonds (29). Two nonviable viral mutants harboring double cysteine mutations in Vp1 (i.e., C49A-C87A and C87A-C254A) fail to traffic to the nucleus but accumulate, at reduced levels, as punctuate speckles in the cytoplasm (30). This finding suggests that the disulfide redox, Vp1 folding, and cytoplasmicnuclear trafficking of Vp1 are all tightly linked (30).Protein folding is generally assisted by molecular chaperones involving transiently and/or partially unfolded proteins (reviewed in reference 37). In particular, Hsc70 and Hsp70, members of the HSP70 family, have been implicated in the life cycle of polyomaviruses (37). Infection by SV40 or murine polyomavirus induces higher levels of production of stress-inducible Hsp70 and/or its homologs (26,27,53). The in vivo association of the constitutively expressed protein Hsc70 with Vp1 proteins of SV40 and murine polyomavirus has also been reported (8,43). The HSP70 chaperones use ATP hydrolysis cycles to toggle between two states: binding to (ADP-bound state) and release from (ATP-b...

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

“…This construction was accomplished by first generating two PCR fragments, with pCI-Vp1⌬C as a template, in two separate rounds of amplification. Note that we used silent unique restriction sites in pCIVp1⌬C: XbaI, AvrII, and XmaI sites located upstream of the Vp1 initiating methionine; an NheI site located within the Vp1 coding sequence (aa 303); and a BamHI site located downstream of H6 (12). The first round of amplification (PCR1) generated an XbaI-to-AvrII fragment that includes the corresponding region in pCI-Vp1⌬C but adds a BsrGI site and the FLAG coding sequence downstream of AvrII (sense primer, 5=-GGT GGG AGG TCT ATA TAA GCA GA; antisense primer, 5=-CTT GTC ATC GTC GTC CTT GTA GTC CAT TGT ACA CCT AGG CGG CCG CGT GCA).…”

“…The construction of pCI-Vp1-⌬C58-H6 (pCI-Vp1⌬C) and its mutant derivatives pCI-Vp1-⌬N20⌬C58-H6, pCI-Vp1⌬C58-H6-C49A-C87A, and pCIVp1⌬C58-H6-C87A-C254A was described previously (10,12). pCIVp1⌬C encodes SV40 Vp1⌬C, which contains Vp1 amino acids (aa) 1 to 298 plus additional amino acids before (MKM) and after it (in order, GPAS, three repeats of the flexible linker GGGGS, and EFESGR) and a His tag (HHHHHH) at the very C terminus.…”

“…U2OS and U2OS/T cells were cultured under conditions identical to those for COS-7 cells. DNA transfection was performed as described previously (12), using 10 g of plasmid DNA per 10-cm plate containing 4 ϫ 10 6 CV1, COS-7, U2OS, or U2OS/T cells or 3 ϫ 10 7 293H, 293T, or 293TT cells. For metabolic radiolabeling, cells transfected with pCI-Vp1⌬C were labeled with 0.4 mCi of [ 35 S]methionine per 10-cm plate at 20 h posttransfection (p.t.…”

“…These species, found in the cytoplasm of COS-7 cells expressing a recombinant Vp1 lacking the C-terminal arm, Vp1⌬C (10), have SDS-PAGE mobilities slower than that of the Vp1 monomer but, unlike the disulfide-containing intermediates, are nonreducible (12). Hence, the mdVp1s appear to harbor unidentified covalent modifications.…”

“…In fact, the mutation of two Vp1 cysteine pairs (C49A-C87A and C87A-C254A) leads to defective Vp1 folding in the cytoplasm and the loss of viral viability (10,11). The mutant Vp1s, despite harboring a normal NLS, are largely blocked in their movement to the cell nucleus and induce an aberrant subcellular localization of the heat shock proteins Hsp70 and Hsc70 (which are referred to as HSP70 here) (10,12).…”