The goal of the studies described in this proposal is to elucidate the mechanism by which gag (core) proteins of the human immunodeficiency virus (HIV- 1) are transported to the plasma membrane of infected cells and assemble into virus particles. Our proposal is of direct medical significance because it focuses of a potentially inhibitable step in the life cycle of a lethal human pathogen. In particular, examination of the HIV gag transport/ assembly process may reveal virus specific steps in transport, membrane binding, and budding which may be blocked without significantly perturbing essential cellular processes. Additionally, our proposal addresses basic unresolved questions concerning enveloped animal virus assembly, targeting of myristylated proteins, peripheral membrane protein interactions, and virus budding. The methods we developed for the study of murine retrovirus assembly will be used in our analysis of HIV.
Our specific aims are as follows: 1. Generation of HIV-1 gag mutants: A limited number of HIV gag protein deletion, insertion, and point mutants will be constructed for analysis in a modified wild type (wt) HIV backbone. We will focus on matrix (MA), capsid (CA), and p6 domains with the intention of identifying mutants involved in the assembly process.2. Screening of mutants for replication defective phenotypes: Gag mutants in a HIV construct carrying a selectable marker will be characterized following transfection into primate cells. Phenotypically wt constructs will be identified by scoring transduction of the selectable marker to human target cells. Assembly mutants will be identified by screening for intracellular p55 gag, and examination of released particles by immunoblotting, reverse transcriptase (RT) assay, electron microscopy (EM), and viral RNA analysis. 3.Characterization of assembly mutants. Gag mutants which are defective in virus assembly will be characterized in detail. Subcellular localization studies of HIV wt and mutant gag proteins will employ fractionation and immunofluorescence protocols. Intracellular particle formation will be monitored by EM, and fractionation in the presence of nonionic detergent. With a limited number of mutants, isolation and mapping of second site revertants will be attempted.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA047088-04A1
Application #
3190562
Study Section
AIDS and Related Research Study Section 3 (ARRC)
Project Start
1991-07-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
4
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Oregon Health and Science University
Department
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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