Little is known about the process through which viral proteins and RNA assemble on the plasma membranes of host cells. The investigators have been studying this process in vitro and find that these complexes can be reconstituted to give particles that resemble immature virion assemblies morphologically and functionally, and thus provide a way to study, at the molecular level, the assembly pathway leading to infectious particle formation. In this proposal, they will test a model for viral assembly that involves initial membrane interactions involving several domains of Gag, that are replaced with protein-protein and protein-RNA interactions as assembly proceeds. They propose that myristylation plays a role in orienting viral Gag on the membrane surface. Further, they propose that a highly conserved region in the capsid domain transduces the conformational changes that occur upon membrane association and find that mutations in this region severely impair assembly. Processing of Gag releases MA causing altered protein contacts mediated through a particular portion of the protein. This serves to weaken MA interaction with the membrane surface, thus allow it to be transported to the host cell nucleus during a new round of infection. To identify properties of the nucleoprotein complexes that might be targeted for design of anti-viral agents, Dr. Scarlata proposes using Gag, the viral protease, and RNA containing the encapsidation signal in the 5' end of the viral genome to define the macromolecular interactions of functional reconstituted nucleocapsids. The applicant will first model by characterizing the in vitro assembly of wild type Gag in terms of conformation, orientation, and role of myristylation using fluorescence energy transfer; nucleoprotein assembly will be monitored by gel-shift, centrifugation, and protection from nuclease degradation; functional competence will be assessed by PR assay; and limited proteolysis and hydrolysis with nucleases will localize altered regions in Gag and RNA, respectively. Second, the applicant will compare the assembly of Gag proteins with mutations in this highly conserved region. Third, the applicant will examine the role of a region of MA in weakening membrane interactions. The proposed study makes it possible to identify critical interactions required for assembly and to design assembly inhibitors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM058271-01
Application #
2698727
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1998-05-01
Project End
2002-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Physiology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Bon Homme, Marjorie; Carter, Carol; Scarlata, Suzanne (2005) The cysteine residues of HIV-1 capsid regulate oligomerization and cyclophilin A-induced changes. Biophys J 88:2078-88
Scarlata, Suzanne; Carter, Carol (2003) Role of HIV-1 Gag domains in viral assembly. Biochim Biophys Acta 1614:62-72
Bouamr, Fadila; Scarlata, Suzanne; Carter, Carol (2003) Role of myristylation in HIV-1 Gag assembly. Biochemistry 42:6408-17
Bon Homme, Marjorie; Wong, Stanislaus; Carter, Carol et al. (2003) The pH dependence of HIV-1 capsid assembly and its interaction with cyclophilin A. Biophys Chem 105:67-77
VerPlank, L; Bouamr, F; LaGrassa, T J et al. (2001) Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci U S A 98:7724-9
Provitera, P; Goff, A; Harenberg, A et al. (2001) Role of the major homology region in assembly of HIV-1 Gag. Biochemistry 40:5565-72
Kikonyogo, A; Bouamr, F; Vana, M L et al. (2001) Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells. Proc Natl Acad Sci U S A 98:11199-204
Ehrlich, L S; Liu, T; Scarlata, S et al. (2001) HIV-1 capsid protein forms spherical (immature-like) and tubular (mature-like) particles in vitro: structure switching by pH-induced conformational changes. Biophys J 81:586-94
Provitera, P; Bouamr, F; Murray, D et al. (2000) Binding of equine infectious anemia virus matrix protein to membrane bilayers involves multiple interactions. J Mol Biol 296:887-98