The overall goal of the research proposed in this renewal application is to understand how HIV-1 Gag is trafficked through the cell, targeted to membranes and assembled into virions. During the previous granting period, we established that HIV-1 assembly is an ordered process that proceeds through sequential intermediates. We showed that Gag is localized to the plasma membrane and that targeting of Gag to membrane raft-like domains, """"""""barges"""""""" promotes efficient particle formation. In addition, we identified a motif (IL) within Gag that regulates its association with multivesicular bodies (MVBs). In the next renewal period, we plan to extend these studies by elucidating the mechanisms for Gag trafficking, subcellular localization, and interaction with the cellular endocytic and exocytic pathways. The proposed research is designed to: 1. Determine the mechanisms that regulate Gag targeting to the plasma membrane vs MVBs in different cell types. We will test the following hypotheses: A) MVB formation is necessaryfor Gag mediated assembly; B) Rab27a regulates exocytosis of Gag-containing MVBs and/or secretory lysosomes. C) Ca++ flux regulates exocytosis of Gag-containing MVBs. In addition, D) We will use TIR-FM (total internal reflection fluorescence microscopy) to visualize and quantitate Gag mediated exocytic events occurring within 100 nm of the plasma membrane. 2. Determine whether Gag expression alters the flux of cellular proteins through the MVB pathway. We will determine whether Gag alters the functioning of the cellular multivesicular body pathway by examining the kinetics of EGF Receptor downregulation and signaling in Gag transfected cells, in pHXB2 transfected cells, and in HIV-1 infected cells. In addition, we will determine whether Gag alters downregulation and signaling via the chemokine and HIV-1 co-receptor, CXCR4, in T cells. 3. Define the mechanisms of Gag membrane targeting, trafficking and assembly by imaging in live and fixed cells. Inducible methods for Gag expression will be exploited to perform time lapse imaging of intracellular trafficking in real time. Multicolor imaging with site-specific fluorescein derivatives (FLASH and ReAsH) will be used to determine the order in which Gag assembly sites are formed and maintained in the cell.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA072309-11S1
Application #
7561399
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Ogunbiyi, Peter
Project Start
1996-07-15
Project End
2010-01-31
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
11
Fiscal Year
2008
Total Cost
$66,530
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Resh, Marilyn D (2012) Targeting protein lipidation in disease. Trends Mol Med 18:206-14
Rai, Tia; Mosoian, Arevik; Resh, Marilyn D (2010) Annexin 2 is not required for human immunodeficiency virus type 1 particle production but plays a cell type-dependent role in regulating infectivity. J Virol 84:9783-92
Martinez, Nathaniel W; Xue, Xiaoxiao; Berro, Reem G et al. (2008) Kinesin KIF4 regulates intracellular trafficking and stability of the human immunodeficiency virus type 1 Gag polyprotein. J Virol 82:9937-50
Valiathan, Rajeshwari R; Resh, Marilyn D (2008) Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag. Virol J 5:23
Resh, Marilyn D (2006) Trafficking and signaling by fatty-acylated and prenylated proteins. Nat Chem Biol 2:584-90
Perlman, Mira; Resh, Marilyn D (2006) Identification of an intracellular trafficking and assembly pathway for HIV-1 gag. Traffic 7:731-45
Resh, Marilyn D (2006) Use of analogs and inhibitors to study the functional significance of protein palmitoylation. Methods 40:191-7
Resh, Marilyn D (2005) Intracellular trafficking of HIV-1 Gag: how Gag interacts with cell membranes and makes viral particles. AIDS Rev 7:84-91
Lindwasser, O Wolf; Resh, Marilyn D (2004) Human immunodeficiency virus type 1 Gag contains a dileucine-like motif that regulates association with multivesicular bodies. J Virol 78:6013-23
Valiathan, Rajeshwari R; Resh, Marilyn D (2004) Expression of human immunodeficiency virus type 1 gag modulates ligand-induced downregulation of EGF receptor. J Virol 78:12386-94

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