This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In comparison to larger DNA viruses, the human immunodeficiency virus (HIV-1) has a relatively limited repertoire of encoded proteins. Given this fact, it is reasonable to expect that the host cell constitutes a rich source of factors that the virus must draw upon for its replication. To date, however, only a few such virus-assisting host proteins have been identified. In this proposal, we endeavor to identify the factors that interact directly with the HIV-1 machinery during viral replication using a system in which viruses have been molecularly engineered to incorporate a potent immunological or biochemical tag. Using this panel of independently tagged replication-competent 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 self-selecting process based on replication competence in culture, the tagged viral proteins must undergo the same interactions encountered by the wild type virus. Therefore, 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. Currently, mass spectrometry techniques is being employed to determine the identity of host proteins and complexes that are captured via their interaction with the tagged viral proteins.
The specific aims of this study are as follows: I. Comprehensive Mutagenesis of the HIV-1 Genome and Selective Recovery of Infectious, Replication-Competent, Tagged Viruses II. Utilization of Tagged Viruses for the Quantitative Recovery of Host Proteins that Interact with Viral Proteins during Replication III. Identification of Interacting Proteins by Mass Spectrometry and Assessment of Their Role in the HIV-1 Infectious Cycle A manuscript describing this work is in preparation
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