A Genetic/Proteomic Approach to Virus-Host Interactions

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DESCRIPTION (provided by applicant): HIV-1 infection depends on both viral and host factors. Given the limited genetic capacity of the virus and complex molecular events during its infection, HIV-1 must interact with an extensive variety of cellular factors to complete its passage through the cell. To date, however, only a few virus-assisting host proteins have been identified. In this proposal, we outline a strategy to elucidate the cellular binding partners that HIV commandeers to infect its target cells using a system in which viruses have been molecularly engineered to incorporate a potent immunological or biochemical tag. Using this panel of independently tagged (3X FLAG epitope or a biotinylation signal peptide, SNAGG), replicationcompetent derivatives, we seek to recover host proteins that interact specifically with the virus as it progresses through its natural life cycle. As these engineered viruses were generated through a process based on replication competence in culture, the tagged integrase protein likely undergo the same interactions encountered by the wild type protein. We believe that this system will afford us a more authentic view of both transient and stable molecular interactions that form during the normal course of HIV infection than other traditional procedures. Initially, we are focusing specifically on the viral integrase protein-a protein involved in such disparate roles as reverse transcription, the nuclear import of viral DNA and its subsequent integration into host chromosomal DNA. This targeted proteomic study will utilize affinity purification coupled with mass spectrometry to determine the identity of host proteins and complexes that are captured via their interaction with the tagged viral proteins. Elucidation of the complex interactions between cellular and viral proteins during HIV infection would be a great asset not only for the detailed insight into retroviral biology but also for the novel targets for antiviral interventions it would unveil. The viral integrase protein of the Human Immunodeficiency Virus (HIV-1) is absolutely required for viral propagation. In addition to its other functions, integrase is known to play a key catalytic role in coordinating the fusion of the genetic information of the virus with that of the host's cell, thereby making the HIV genome a permanent fixture in the cells of an infected individual. However, most of the biology we understand about the molecular events in HIV infection is from the perspective of the virus, and not from that of the host cell with which it must associate intimately to produce progeny. Thus, this proposal describes methodology that strives to identify the cellular protagonists of the viral integrase; the factors that integrase must rely on for its diverse functions. Because the viral-host interface remains largely unexplored as a target for antiretroviral therapy, inhibitors of their association would represent a new class of antiretroviral agents not previously appreciated.