The HIV capsid (CA) protein appears to be essential to particle assembly, incorporation of HIV pol gene products into particles, and formation of the mature virus core. The investigators will use biophysical, biochemical and genetic methods to examine structural interactions of HIV core proteins, to test structural predictions, and to develop in vitro oligomerization protocols for the assay of potential peptide-based methods for interference of Gag function. Sp.
Aim 1. Electron diffraction-image analysis of the HIV core proteins: Purified histidine-tagged (his- tagged) HIV CA and Gag protein derivatives will be used to make two dimensional protein arrays on phosphatidyl choline (PC) monolayers containing novel nickel-chelating lipids. Stained protein arrays will be viewed by transmission electron microscopy (EM) to obtain data concerning capsid-capsid interactions. Unstained samples will be analyzed to obtain 5-10 angstrom resolution maps by image enhancement- electron diffraction methods. Sp.
Aim 2. Examination of Gag interactions in vitro: Gag-Gag protein association will be assayed in vitro to determine optimal oligomerization conditions, and potential capsid-derived peptide inhibitors of assembly will be tested in the in vitro system. Epitope library screens also will be employed for identification of peptides which bind CA and may serve as candidate peptide inhibitors of core protein functions. Sp.
Aim 3. Genetic and biochemical analysis of HIV virions: Genetic interactions of the capsid domain will be investigated via complementation analysis, and by assay for dominant-negative phenotypes to locate potential sites for antiviral interference. Putative capsid-binding peptides, identified in in vitro studies, will be tested for assembly into virus particles and inhibition of viral replication, and models of Gag protein interactions in virus particles will be evaluated.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA047088-09
Application #
2390700
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1991-07-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
9
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
009584210
City
Portland
State
OR
Country
United States
Zip Code
97239
Clish, C B; Peyton, D H; Barklis, E (1998) Solution structures of human immunodeficiency virus type 1 (HIV-1) and moloney murine leukemia virus (MoMLV) capsid protein major-homology-region peptide analogs by NMR spectroscopy. Eur J Biochem 257:69-77
Zhang, Y; Qian, H; Love, Z et al. (1998) Analysis of the assembly function of the human immunodeficiency virus type 1 gag protein nucleocapsid domain. J Virol 72:1782-9
Barklis, E; McDermott, J; Wilkens, S et al. (1998) Organization of HIV-1 capsid proteins on a lipid monolayer. J Biol Chem 273:7177-80
Barklis, E; McDermott, J; Wilkens, S et al. (1997) Structural analysis of membrane-bound retrovirus capsid proteins. EMBO J 16:1199-213
Zhang, Y; Barklis, E (1997) Effects of nucleocapsid mutations on human immunodeficiency virus assembly and RNA encapsidation. J Virol 71:6765-76
Birrell, G B; Hedberg, K K; Barklis, E et al. (1997) Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C. J Cell Biochem 65:550-64
McDermott, J; Farrell, L; Ross, R et al. (1996) Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents. J Virol 70:5106-14
Hansen, M S; Barklis, E (1995) Structural interactions between retroviral Gag proteins examined by cysteine cross-linking. J Virol 69:1150-9
Wang, C T; Barklis, E (1993) Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants. J Virol 67:4264-73
Wang, C T; Zhang, Y; McDermott, J et al. (1993) Conditional infectivity of a human immunodeficiency virus matrix domain deletion mutant. J Virol 67:7067-76

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