ANALYSIS OF INTEGRASE IN REVERSE TRANSCRIPTION

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There remains a large gap in our understanding of the early events of the HIV-1 virus life cycle. This is due in part to the manner in which HIV-1 proteins are encoded and assembled into the virus particle. The retroviral enzymes are encoded within the C- terminal Pol component of the Gag-Pol precursor polyprotein. All retroviruses encode at least for enzyme activities: protease (PR), polymerase and ribonuclease H (RNase H) activities of the reverse transcriptase (RT), and integrase (IN). HIV-1 encodes and incorporates PR, RT and IN as the Pol components of a 160 kDA Gag-Pol precursor (Pr160Gag/Pol). Mutations within Gag-Pol are pleiotropic, since they can affect the precursor protein during assembly and maturation (late stage), as well as the functions of the mature Gag (MA, CA, NC, p6) and Pol (PR, RT, IN) proteins, after proteolytic processing and virus infection (early events). For example, mutations in In have been shown to alter virus replication through various mechanisms and at different stages in the virus life cycle. This likely explains the diverse phenotypes that have been reported for IN mutant viruses, including those with defects in assembly, virion morphology, reverse transcription, nuclear import, and integration of theprovirus. Recently, we analyzed the function of the HIV-1 IN protein dependently of its expression as part of Gag-Pol, via expression in trans as a Vpr-IN fusion protein. Our analysis has revealed that the mature IN protein itself is required for efficient reverse transcription in vivo. Our data and others also indicate that the DNA synthesis defect is at or prior to initiation and elongation, since the IN mutants synthesize an equal amount of early and late DNA, albeit significantly less than that of wild-type virus. These findings demonstrated that we can, for the first time, uncouple the analysis of IN in early and late events, in the context of a replicating virus (in vivo). The central hypothesis of this application is that the IN protein plays an important role in the initiation stage of reverse transcription through interactions with other viral components of the reverse transcription complex within the infected cell. Further analysis is necessary to understand the mechanisms by which IN augments viral DNA synthesis. The specific objectives of this proposal are: (1) to define the stage in the virus life cycle that IN promotes RT; (2) to define specific viral components that affect the ability of IN to augment RT; (3) to determine whether other retroviral IN proteins are required for efficient RT; and (4) to map the determinants of the IN protein that are required for DNA synthesis. These studies will help to define the IN protein's role in HIV-1 RT in the context of the nucleoprotein complex, and understand the specific molecular interactions between the IN protein and other components of the nucleoprotein complex that required for efficient RT in the infected cell.