Nucleoside reverse transcriptase (RT) inhibitors play an important role in therapy for HIV-1 infection and AIDS; however, long-term use of these drugs is limited by the selection of resistant mutants. These compounds give rise to chain-terminating nucleotides that are incorporated by HIV-1 RT and block further DNA chain elongation. Work from this laboratory was instrumental in showing that HIV-1 RT can catalyze the excision in vitro of chain-terminating nucleotides by transfer to a nucleotide acceptor and that a major class of nucleoside resistant mutants of HIV-1 encodes RT with increased nucleotide-dependent excision activity. Research is proposed to (a) define factors that determine the efficiency of the excision reaction in vitro including structural features of the acceptor substrate, structure of the chain-terminating residue, and sequence elements in the primer-template, (b) investigate the mechanism of inhibition of excision and DNA synthesis by foscarnet (PFA), a structural analog of pyrophosphate, and to determine the mechanism of resistance to PFA by PFA-resistant mutants, (c) investigate the effect of non-nucleoside RT inhibitors on excision and on dNTP inhibition of excision, and (d) to identify factors that determine which intracellular compounds serve as acceptor substrates for excision in vivo -- i.e., in lymphocytes from uninfected and HIV-1 infected individuals. In addition, research is proposed to extend these studies to identify factors that control excision in vivo using an enzymatic assay to detect the products of excision. The goal of this research is to define the role of the excision activity in HIV-1 infection, which will be crucial for the development of more effective drugs and therapeutic strategies against HIV, particularly against the emergence of resistant virus.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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AIDS Discovery and Development of Therapeutics Study Section (ADDT)
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Ussery, Michael A
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University of Miami School of Medicine
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Lu, Gaofei; Matsuura, Suzanne E; Barrientos, Antoni et al. (2013) HIV-1 infection is blocked at an early stage in cells devoid of mitochondrial DNA. PLoS One 8:e78035
Acosta-Hoyos, Antonio J; Matsuura, Suzanne E; Meyer, Peter R et al. (2012) A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation. J Virol 86:5122-33
Scott, Walter A (2011) Structures of reverse transcriptase pre- and post-excision complexes shed new light on HIV-1 AZT resistance. Viruses 3:20-5
Acosta-Hoyos, Antonio J; Scott, Walter A (2010) The Role of Nucleotide Excision by Reverse Transcriptase in HIV Drug Resistance. Viruses 2:372-394
Rutvisuttinunt, Wiriya; Meyer, Peter R; Scott, Walter A (2008) Interactions between HIV-1 reverse transcriptase and the downstream template strand in stable complexes with primer-template. PLoS One 3:e3561
Meyer, Peter R; Rutvisuttinunt, Wiriya; Matsuura, Suzanne E et al. (2007) Stable complexes formed by HIV-1 reverse transcriptase at distinct positions on the primer-template controlled by binding deoxynucleoside triphosphates or foscarnet. J Mol Biol 369:41-54
Meyer, Peter R; Smith, Anthony J; Matsuura, Suzanne E et al. (2006) Chain-terminating dinucleoside tetraphosphates are substrates for DNA polymerization by human immunodeficiency virus type 1 reverse transcriptase with increased activity against thymidine analogue-resistant mutants. Antimicrob Agents Chemother 50:3607-14
Smith, Anthony James; Scott, Walter Alvin (2006) The influence of natural substrates and inhibitors on the nucleotide-dependent excision activity of HIV-1 reverse transcriptase in the infected cell. Curr Pharm Des 12:1827-41
Smith, Anthony J; Meyer, Peter R; Asthana, Deshratn et al. (2005) Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations. Antimicrob Agents Chemother 49:1761-9
Meyer, Peter R; Smith, Anthony J; Matsuura, Suzanne E et al. (2004) Effects of primer-template sequence on ATP-dependent removal of chain-terminating nucleotide analogues by HIV-1 reverse transcriptase. J Biol Chem 279:45389-98

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