Like all retroviruses, it is necessary for HIV to convert its genomic RNA into double stranded DNA. This process of reverse transcription is catalyzed by the reverse transcriptase (RT). The HIV-I RT protein has multiple functions and requires interaction with several viral and cellular proteins within the virion and the infected host cell that are necessary for complete synthesis of the viral DNA. In most RT studies bacterially expressed RT protein has been used for secondary structure analysis, to identify subfunctional domains and to help understand structure-function relationships. The results of these efforts have contributed greatly to the current understanding of reverse transcription in vitro, at the molecular level. Recent studies suggest that reverse transcription in the infected cell (in vivo) is more complex than was expected because proviral DNA synthesis in infected cells requires other viral and cellular proteins. Currently there are not good systems available to study reverse transcription in vivo, in part, because of inherent difficulties in manipulating RT expression and incorporation independent of gag/pol. Our recent studies on HIV accessory proteins have shown they can be exploited as vehicles to incorporate functional proteins into virions by expression in trans as heterologous fusion molecules. We have shown that the protease (PR-mutant) integrase (IN), and reverse transcriptase (RT) can be incorporated into virions by expression in trans as Vpr-fusion proteins. Importantly, our preliminary data demonstrate that the DNA synthesis of IN and RT defective proviruses can be restored by trans complementation with Vpr-RT and Vpr-IN fusion proteins, respectively. In this application, we proposes to exploit Vpr (and possibly Gag) as a vehicle to incorporate functional RT protein into virions to establish a model system that will facilitate studies on the processes of retroviral reverse transcription in infected cells. The central hypothesis of this project is that, by defining the key components necessary for efficient trans complementation of RT function, we can develop a model system to study processes of reverse transcription in a biologic relevant context. In the application, we propose to construct an RT defective backbone provirus, which can be effectively rescued (for DNA synthesis) by trans complementation with an RT fusion protein whose function mimics that of the virus- encoded RT. To achieve these goals we propose: i. To determine the role of the RT coding sequence in Gag/Pol expression, and proteolytic processing of the Gag protein; 2. To analyze the effects of RT mutants encoded by the provirus on the function of the Vpr-RT fusion protein; 3. To analyze the effect of the RT fusion partner on trans complementation of RT defective HIV-I; 4. To analyze RT defective virus replication by trans complementation using stable cell lines; and 5. To investigate the role of IN reverse transcription in infected cells. Study of HIV-I reverse transcription in infected cell will help to understand the precise molecular mechanisms of provirus DNA synthesis and facilitate development of novel antiretroviral strategies.
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