The current application explores different approaches to related questions about retrovirus assembly and maturation. The laboratory of PI has established a research program studying the assembly and maturation of a primate D-type retrovirus, Mason-Pfizer Monkey Virus (M-PMV). The laboratory of Dr. Ruml has established a method for efficient expression of retroviral structural proteins in S. cerevisiae and E. coli.. This ability to assemble capsids from bacterially-expressed protein allows important questions on the mechanism of assembly and proteolytic cleavage of M-PMV polyprotein precursors to be addressed. The M-PMV proteinase has been purified and initially characterized in the group of Dr. Pichova and this capability will provide the basis for structural, biochemical and genetic studies of enzyme function. Specifically we propose to: 1. Investigate and optimize the conditions for efficient in vitro assembly of retroviral capsids from bacterially expressed precursors. 2. Characterize the structure of the in vitro assembled capsids. 3. Determine regions of the Gag related precursors responsible for assembly by evaluation of assembly competence of mutants in the regions which are not assembled in mammalian cells. The in vitro system will eliminate the effect of cellular factors on assembly. 4. Investigate the effect of different ratios of Gag related polyproteins on in vitro co-assembly of immature capsids and co-assembly of homologous precursors from different retroviruses (particularly D- with C-types) into one immature capsid. 5. Investigate the localization of cleavage sites in the immature capsid by the evaluation of proteolysis of different cleavage sites in the immature capsid using mutant Gag precursors. 6. Analyze the structure/function relation of the M-PMV proteinase and evaluate the roles of 26 kDa, 17 kDa and 12 kDa forms, which we have recently purified.
| Pereira, Lara E; Clark, Jasmine; Grznarova, Petra et al. (2014) Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules. Virology 449:109-19 |
