The role of Hsp70 chaperones in papovavirus disassembly and assembly

• Main Author: Chromy, Laura R
• Format: Dissertation
• Language: English
• Subjects: molecular biology; virology
• ISBN: 0549033025; 9780549033028

Papovaviruses include the small DNA tumor virus families Polyomaviridae and Papillomaviridae. Although the families are classified separately based on genome organization and pathogenesis, these viruses share structural characteristics. The non-enveloped papovavirus capsid consists of 72 pentamers arranged on a T=7 icosahedral lattice. Each pentamer contains 5 copies of the major capsid protein, VP1 for polyoma and L1 for papilloma. VP1 and L1 pentamers have nuclear localization signals that bind karyopherins for nuclear transport and bind DNA non-specifically. Purified recombinant pentamers of either VP1 or L1 readily self-assemble into capsids in vitro under appropriate buffer conditions. Thus, unknown cellular factors regulate both capsid disassembly during the early phases of virus infection and exclusive encapsidation of the viral genome during assembly of virus progeny.
Prior results found cellular Hsp70 family chaperones associated with VP1 pentamers in a spatial and temporal manner consistent with binding during translation until delivery to the nucleus. As expected from previously described roles for chaperones, Hsp70 family chaperones were found to be important for proper capsid protein folding in this study. Hsp70 chaperones were also found to primarily interact with the C-terminal domain of polyoma VP1 that forms interpentameric contacts in the assembled capsid. Hsp70 chaperone protein bound to VP1 prevented capsid assembly, but chaperones did not prevent interactions with DNA. These results suggested an active role for chaperones in regulating pentamer to capsid conversions. Using in vitro reactions that required ATP, Hsp70 family chaperones were found to disassemble both polyoma and papilloma virus-like particles, as well as intact virions. In the reverse reaction, the Hsp70 bacterial chaperone DnaK, assembled pentamers into uniform capsids, in reactions dependent upon ATP hydrolysis and the co-chaperones DnaJ and GrpE. The eukaryotic chaperone Hsc70 was also found to assemble VP1 pentamers when stimulated by the J-domain of large T-antigen, the viral protein that replicates the viral genome. These findings lead to the hypothesis that Hsp70 chaperone families participate in both papovavirus disassembly and assembly, and that the direction of the equilibrium between these reactions in the cell could depend on co-chaperones or other regulatory cofactors found in different cellular compartments.