The overall objective of this research proposal is to define at the molecular level the individual steps in reverse transcription of the HIV-l viral genome. The experimental approach proposed here is unique because it uses viruses derived from mutant proviral genomes to study reverse transcription. Three key areas of reverse transcription, for which there is very little information, will be investigated. First, the isolated virions from HIV-l infected cells contain the necessary viral proteins for reverse transcription. How the virus assembles these components into the virion remains a major question. Second, the initiation of reverse transcription requires a cellular tRNA-Lys as aprimer and occurs at a region of the viral RNA genome, the primer binding site (PBS). How the viral proteins recognize the PBS and how the tRNA-Lys is selected for inclusion into the virion is unknown. Third, the process of reverse transcription requires template switching by the reverse transcriptase. How this occurs at the molecular level is not understood. To address these questions, the following specific aims are proposed: 1. To define the roles of the Gag-Pol polyprotein in reverse transcription. The reverse transcriptase is incorporated into the virion in the form of a larger precursor molecule, pr160(gag-Pol). The regions of Gag-Pol which are required for incorporation into virions, and the interaction between Gag-Pol and the cellular tRNA-Lys will be analyzed using a complementation system developed by this laboratory. We have demonstrated that the unprocessed Gag-Pol polyprotein contains reverse transcriptase activity and the function of this activity in initiation of reverse transcription will be investigated. 2. To determine the structural features of the viral RNA genome containing the PBS required for initiation of reverse transcription. The minimum number of nucleotides within the PBS and the region of the viral RNA genome encompassing the PBS which is required for initiation of reverse transcription will be determined. 3. To characterize features of the viral genome required for template switching in reverse transcription. Mutations in the PBS will be constructed to investigate the requirements for template switching. In addition, the role of reverse transcriptase in promoting template switching will be analyzed. Reverse transcription is an early step in viral replication and is a major target for anti-viral drugs. The long-term goal of this research is to delineate the in vivo details of reverse transcription which will provide new targets for therapeutic strategies to inhibit this critical step in HIV-l replication.
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