HIV-1 is the causative agent of AIDS. Three viral enzymes - RT, integrase (IN), and protease (PR) - have essential roles in the replication of HIV-1. We are engaged in a long-term effort to study HIV-1 RT, with the expectation that this information will be useful in the development of more effective anti-RT drugs. Our strategy has involved the synthesis, using recombinant DNA techniques, of both wild-type and mutant HIV-1 RTs, including drug-resistant mutants. Some of this purified RT has been used by our long-term collaborator, Edward Arnold (Rutgers University), for structural studies. We have used purified HIV-1 RT to study the biochemical properties of RT mutants, including drug-resistant mutants, and in some cases, have compared the properties of HIV-1 RT to other retroviral RTs, primarily murine leukemia virus (MLV) RT. A major focus of our recent work on HIV-1 RT is the mechanism(s) of nucleoside RT inhibitor (NRTI) resistance. NRTIs are widely used to treat HIV-1 infections; however, there are serious problems with drug toxicity and with the development of resistance. Two of the commonly used NRTIs, 3TC and AZT, have elements (""""""""handles"""""""") that project beyond the corresponding positions of normal dNTPs. 3TC- and AZT-resistant HIV-1 RTs take advantage of these handles and use steric hindrance to block the incorporation of 3TC and to enhance the excision of AZT. Unfortunately, there are HIV-1 RTs that are resistant to NRTIs that do not have obvious handles. We are investigating the mechanisms that underlie drug resistance - in particular, we want to understand the mechanisms that underlie resistance to multiple NRTIs. We have found that the newly approved drug, tenofovir, is efficiently excised by RTs that can excise a number of other NRTIs. We have started a collaboration with a skilled nucleoside chemist, Victor Marquez (Laboratory of Medicinal Chemistry, NCI); we are investigating whether it is possible to develop a class of nucleoside analogs that are relatively resistant to excision by HIV-1 RTs that can excise multiple NRTIs. All of these experiments involve a combined structural/biochemical approach; what we have learned from RT structure has been an invaluable guide for planning the biochemical studies, while the results and hypothesis generated in the biochemical experiments have inspired new rounds of structural experiments. NRTI resistance is only one aspect of the behavior of RT. We also want to understand how RT carries out reverse transcription in an infected cell, and to correlate the wealth of structural and biochemical data on HIV-1 RT with the actual process of reverse transcription. These experiments are part of Project Z01 CCR 010482 (Retroviral Replication and Vector Design). In some cases, it will not be possible to explain the in vivo data with the available structural and biochemical results. In such cases, we will do additional biochemical and structural experiments to complement (and better understand) the in vivo results.
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