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

Abstract

The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1⌬C, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1⌬C in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1⌬C. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins.

Inhibition of large T antigen ATPase activity as a potential strategy to develop anti-polyomavirus JC drugs

“…Therefore, inhibitors of Hsp70 would be expected to curtail cell growth as well as polyomavirus replication by creating a less favorable milieu for the virus to thrive, and perhaps even causing selective apoptosis of infected cells (Wright et al, 2008). Moreover, Hsp70 and the cochaperone Hsp40 have been implicated in the correct folding of the SV40 VP-1 capsid protein into functional pentamers (Li et al, 2009; Watanabe et al, 2013). Accordingly, an Hsp70 inhibitor, MAL2-11B, has been shown to cause one log reduction of BKV viral load in HK2 cells at concentrations of 15 μM, and a fivefold reduction in SV40 DNA replication at a concentration of 100 μM (Wright et al, 2009).…”

The cellular model for Alzheimer's disease research: PC12 cells

“…COS-7 cells (ATCC ® CRL-1651™) are transformed with an origin-defective mutant of Simian Virus 40 (SV40) [As a polyomavirus, SV40 has an intricate structure. The capsid is comprised of the major capsid protein VP1 and the minor capsid proteins VP2 and VP3 (Watanabe et al, 2013 )]. The α3 nAChRs, α4 nAChRs and β2 nAChRs have been shown to be expressed on the surface of COS-7 cells (Neff et al, 1995 ).…”