Virus particle assembly of HIV-1, the causative agent of AIDS, takes place at the plasma membrane (PM) in most cell types including natural host T cells. Gag localization to the PM is driven by the matrix (MA) domain. MA mediates membrane binding of Gag via N-terminal myristoyl moiety and a highly basic region (HBR) that binds acidic lipids. Binding of HBR to a PM-specific acidic phospholipid PI(4,5)P2 is critical for PM localization of Gag and efficient virus release. Notably, in vitro studies showed that MA HBR also interacts with RNA, which suppresses binding of Gag to non-PI(4,5)P2 acidic lipids, suggesting that RNA is involved in MA-membrane interactions. However, mechanisms by which PI(4,5)P2 and RNA regulate PM-specific Gag localization remain to be elucidated. Once at the PM, Gag further associates specifically with membrane microdomains known as lipid rafts as well as larger PM domains. In virus-producing T cells contacting uninfected cells, Gag, along with other viral and cellular proteins, specifically accumulates at the area of the PM forming a cell-cell junction known as the virological synapse (VS). The VS facilitates cell-to-cell virus transmission via efficient transfer of newly formed virus particles. Notably, in T cells, Gag multimers localize to a rear-end protrusion termed the uropod that eventually constitutes the VS. Despite the importance in virus spread, however, the mechanism by which Gag multimers localize to uropods and eventually to the VS is not well understood. Our long-term goal is to elucidate mechanisms that determine subcellular sites of HIV-1 assembly. Our central hypothesis in this application is that competition between acidic lipids and RNA for MA binding determines Gag localization to the plasma membrane, where Gag multimerization and microdomain association facilitate Gag accumulation at the uropod that eventually forms virological synapses. To test this hypothesis, we plan to pursue the following three specific aims: [Aim 1] Determine the mechanisms by which lipids and RNA regulate PM binding of Gag. Using in vitro and cell-based assays for Gag-membrane interactions, we will elucidate molecular determinants for the competition between RNA and acidic lipids and its downstream effect on Gag multimerization. [Aim 2] Elucidate the determinants for association between Gag and membrane microdomains. Using a novel in vitro system, we will determine contributions of a unique mode of MA-PI(4,5)P2 interaction and Gag multimerization to Gag-raft association. [Aim 3] Identify the mechanism by which nucleocapsid-driven multimerization directs Gag to the uropod. Using biochemical and high-resolution microscopy methods, we will analyze association of Gag multimers with uropod-directed membrane proteins that might link Gag to rearward actin flow. The knowledge gained from experiments outlined in this proposal will likely help us develop strategies for pharmacological intervention of mechanisms regulating Gag localization to the PM and the VS, thereby inhibiting extracellular virus release and cell-to-cell transmission.

Public Health Relevance

Proper localization of viral components in cells is critical for efficient production of virus particles and the spread from infected to uninfected cells. The goal of the proposed research is to elucidate the mechanisms that direct Gag, a structural protein of HIV-1 that causes AIDS, to the proper sites within virus-expressing cells. Knowledge obtained from the proposed studies will help us to develop novel antiviral strategies that may either directly suppress assembly and release of HIV-1 particles or inhibit the spread of this virus to uninfected host cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI071727-07
Application #
8463448
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sharma, Opendra K
Project Start
2006-07-01
Project End
2017-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
7
Fiscal Year
2013
Total Cost
$353,922
Indirect Cost
$118,922
Name
University of Michigan Ann Arbor
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Inlora, Jingga; Collins, David R; Trubin, Marc E et al. (2014) Membrane binding and subcellular localization of retroviral Gag proteins are differentially regulated by MA interactions with phosphatidylinositol-(4,5)-bisphosphate and RNA. MBio 5:e02202
Chukkapalli, Vineela; Inlora, Jingga; Todd, Gabrielle C et al. (2013) Evidence in support of RNA-mediated inhibition of phosphatidylserine-dependent HIV-1 Gag membrane binding in cells. J Virol 87:7155-9
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Inlora, Jingga; Chukkapalli, Vineela; Derse, David et al. (2011) Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag. J Virol 85:3802-10
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Monde, Kazuaki; Chukkapalli, Vineela; Ono, Akira (2011) Assembly and replication of HIV-1 in T cells with low levels of phosphatidylinositol-(4,5)-bisphosphate. J Virol 85:3584-95
Chukkapalli, Vineela; Ono, Akira (2011) Molecular determinants that regulate plasma membrane association of HIV-1 Gag. J Mol Biol 410:512-24
Ono, Akira (2010) Relationships between plasma membrane microdomains and HIV-1 assembly. Biol Cell 102:335-50
Llewellyn, G Nicholas; Hogue, Ian B; Grover, Jonathan R et al. (2010) Nucleocapsid promotes localization of HIV-1 gag to uropods that participate in virological synapses between T cells. PLoS Pathog 6:e1001167

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