The goal of this proposal is to characterize the molecular interactions involved in the selective and functional incorporation of viral and cellular proteins into virions of HIV-1, the causative agent of AIDS. Our preliminary data show that specific domains of the HIV-1 Gag polyprotein are both required and sufficient for the incorporation of the viral envelope glycoprotein and of the cellular protein cyclophilin A. The interactions that lead to the incorporation of these proteins are critical since the envelope glycoprotein mediates viral entry and cyclophilin A affects the infectivity of HIV-1 virions. Our preliminary data show that the requirements for HIV-1 envelope glycoprotein incorporation, which is governed by the matrix domain of the Gag polyprotein, are unusually stringent. Our data further show that the striking dependence of HIV-1 envelope glycoprotein incorporation on the integrity of the HIV-1 matrix domain arises from the presence of sequences within the cytoplasmic tail of the transmembrane glycoprotein gp41. Our preliminary results demonstrate that the virion-association of cyclophilin A, the major intracellular receptor for the immunosuppressive drug cyclosporin A (CsA), is mediated by the capsid domain of the HIV-1 Gag polyprotein. We also showed that CsA and the non-immunosuppressive analogue SDZ NIM 811, which both bind to cyclophilin A and inhibit its virion-association, reduce the infectivity of HIV-1 virions. Moreover, we found that while SDZ NIM 811 has no effect against the closely related SIVmac, which does not incorporate cyclophilin A, sensitivity to the drug can be transferred onto SlVmac by transfer of the HIV-1 capsid domain. To locate key sequences within the matrix domain for envelope glycoprotein incorporation and determinants within the capsid domain that confer the ability and/or necessity to incorporate cyclophilin A, the properties of mutant and chimeric Gag proteins will be studied. Mutagenesis will also be performed to study the role of the cytoplasmic domain of gp41 in the requirement for matrix domain sequences for incorporation and to identify the virion- association motif(s) in cyclophilin A. Studies to identify the steps in the virus life cycle affected by the Gag-cyclophilin interaction are also proposed. An understanding of the molecular requirements for the association of these viral and cellular proteins with HIV-1 virions could provide specific targets for anti-viral therapy.
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