The proposed studies aim to understand the molecular mechanisms governing assembly and maturation of retroviral capsids, specifically the role of the matrix (MA) protein and protease (PR). Previous studies by the PI with his foreign collaborators, using Mason-Pfizer Monkey virus (M-PMV) as a model system, have shown the assembly of retroviral capsids from the gag precursors in vitro after over expression of this protein in bacteria and a new-form of the proteinase. The proposed studies will extend these observations to identify the domains of gag required for capsid targeting and assembly (specific aim 1) and to study the proteolytic process of the gag polyproteins (specific aim 2). The subgoals for these specific aims are to: la. Clone and express the M- PMV membrane targeting defective MA mutants; 1b. Determine the structure of the mutant MA protein and compare its structure with the wild type protein to elucidate the importance of specific domains in structural changes that affect intracellular targeting, assembly, and transport; 1c. Study the effect of N-terminal myristylation on MA protein structure; 1d. Express and purify p12 and p16 for structural analyses; 1e. Study the role of the MA domain in assembly of immature capsids using chimeric gag genes from C and D-type retroviruses; 1f. Determine the role of the gag-encoded N-terminal domains for efficient assembly of immature capsids; 2a. Study the role of the truncated proteinase species in the viral replication cycle; 2b. Study the substrate specificity of the proteinase using the gag polyprotein; 2c. Screen for inhibitors of the proteinase using a combinatorial peptide library; 2d. Crystallize and determine the structure of the different forms of the proteinase.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW000050-09
Application #
6322351
Study Section
International and Cooperative Projects 1 Study Section (ICP)
Program Officer
Michels, Kathleen M
Project Start
1998-09-30
Project End
2003-10-31
Budget Start
2000-11-01
Budget End
2003-10-31
Support Year
9
Fiscal Year
2001
Total Cost
$61,103
Indirect Cost
Name
University of Alabama Birmingham
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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