The proposed studies are aimed at understanding how the HIV-1 Gag polyprotein and its constituent matrix (MA) and capsid (CA] domains function during viral replication. During the current funding period we identified the structural determinants responsible for targeting HIV-1 Gag to virus assembly sites on the plasma membrane (PM). Specifically, we showed that phosphatidyIinositoI-4,5-bisphosphate (PIP2), a constituent of the PM, binds to a conserved cleft on the HIV-1 MA protein and triggers exposure of the N-terminal myristyl group. The unsaturated 2'-acyl chain of PIP2 is sequestered within the cleft of the protein, and exposure of the saturated 1'-chain and myristyl group explains how HIV-1 Gag is targeted specifically to lipid raft assembly sites. We further showed that conservative mutations that mistarget Gag to the cytoplasm in vivo 'turn off the HIV-1 myristyl switch; that HlV-2 Gag trafficking is also P1P2 dependent; and that the switch behavior of HIV-2 MA is more tightly regulated than that of HIV-1, which could explain the poor replication properties of the virus in some cell types and in humans. In addition, we determined the first atomic level strucuture of an antiviral assembly inhibitor bound to the HIV-1 CA protein. We are now poised to extend studies to macromolecular interacdons that occur during HIV assembly, including interactions among viral constituents and between viral and cellular constituents. A new system for detecting interactions between HIV-1 MA and the intra-viral segment of the envelope glycoprotein GP41 has been developed, which will be used to identify specific interactions and structures responsible for recuriting the envelope protein into assembling particles. Studies of the myristyl switch will be extended to animal viruses commonly used as models for HIV-1 and for drug testing, Including SIV and BLV. New efforts will be made to identify compounds that bind to conserved sites on HlV-1, HIV-2, and SIV MA and inhibit myristate exposure. Finally, based on our high resolution structure of the CA:CAP-1 complex, and as part of a new collaboration with Vironova Pharmaceuticals (Sweden), we intend to rationally design capsid assembly inhibitors with improved antiviral properties.
As HIV continues to evolve, it is increasingly important to develop new therapies that exploit different viral targets. The proposed studies aim to enhance understanding of the molecular structures and mechanisms associated with HIV-1 assembly and maturation and to facilitate the rational design of new therapeutics that inhibit this phase of replication.
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|Tedbury, Philip R; Mercredi, Peter Y; Gaines, Christy R et al. (2015) Elucidating the mechanism by which compensatory mutations rescue an HIV-1 matrix mutant defective for gag membrane targeting and envelope glycoprotein incorporation. J Mol Biol 427:1413-27|
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|Saad, Jamil S; Ablan, Sherimay D; Ghanam, Ruba H et al. (2008) Structure of the myristylated human immunodeficiency virus type 2 matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting. J Mol Biol 382:434-47|
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