The broad objective of this proposal is to elucidate the mechanism of HIV-1 reverse transcription at the molecular level. The reverse transcription of the HIV-1 viral genome is a critical step in the replication cycle of the virus which is only poorly understood. At present, there is no system available for the thorough study of the biochemical processes required for HIV-1 reverse transcription. In order to approach this problem, we plan to express and characterize HIV-1 proteins required for reverse transcription. Specifically, enzymatically active RT from E. coli will be characterized, and techniques for the complete biochemical purification will be developed. Additional virion proteins (eg. gag gene products, endonuclease, accessory gene products) which might also be required in reverse transcription will be expressed using a similar approach. We will develop a reconstituted, in vitro system which accurately reflects HIV-1 reverse transcription. The expressed HIV-1 RT, and template RNA, generated from in vitro transcribed infectious HIV-1 proviral DNA, will be used to develop an in vitro system in which we can define the molecular events of HIV-1 reverse transcription. The specific structural features of HIV-1 RT will be correlated with function in reverse transcription of the HIV-1 genome. DNA mutagenesis will be used to create mutant RTs to elucidate structural features of this protein that are required for activity in the in vitro reverse transcription system. Finally, mutant RT will be tested for the capacity to interfere with reverse transcription in vitro and inhibit HIV-1 replication in susceptible tissue culture cells. An important component in the development of a rational approach for the control of AIDS is to elucidate the events of viral replication. The long term goal of this project, to understand the molecular details of the mechanism of HIV-1 reverse transcription, will provide a foundation for the development of novel methods in which to more effectively control virus replication.
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