Structure and Function of HIV-1 Reverse Transcriptase: Molecular Mechanisms of Polymerization and Inhibition

Christopher Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO 65211, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 12/2008; 385(3):693-713. DOI: 10.1016/j.jmb.2008.10.071
Source: PubMed


The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular, drugs that are effective against the current drug-resistant strains of HIV-1.

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    • "When NRTIs and non-NRTIs are used together, their combinatorial effect can be synergistic in reducing viral load and provide a higher threshold to viral resistance. NNRTIs have been used against HIV for several years and bind to a pocket near the active site (Das et al., 2012; Sarafianos et al., 2009). This allosteric site, when bound to non-NRTIs, prevents the conformational changes required for the HIV RT to affix a new nucleotide. "
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    ABSTRACT: Hepatitis B virus (HBV) infections rely on the proper functioning of the viral polymerase enzyme, a specialized reverse transcriptase (RT) with multiple activities. All currently approved antiviral drugs for the treatment of chronic hepatitis B virus, except for interferon, target the RT and belong to the same chemical class - they are all nucleoside analogs. Viral DNA synthesis is carried out by the RT enzyme in several different steps, each with distinct RT conformational requirements. In principle, each stage may be targeted by distinct antiviral drugs. In particular, the HBV RT has the unique ability to initiate viral DNA synthesis using itself as a protein primer in a novel protein priming reaction. In order to help identify RT inhibitors and study their mechanisms of action, a number of experimental systems have been developed, each varying in its ability to dissect the protein priming and the subsequent stages of viral DNA synthesis reaction at the molecular level. Two of the most effective drugs to date, entecavir and tenofovir, can inhibit both the protein priming and the subsequent DNA elongation stages of HBV DNA synthesis. Interestingly, clevudine, a thymidine analog, can inhibit both protein priming and DNA elongation in a non-competitive manner and without being incorporated into the viral DNA. Thus, a nucleoside RT inhibitor (NRTI) can functionally mimic a non-NRTI (NNRTI) in its inhibition of the HBV RT. Therefore, novel NRTIs as well as NNRTIs may be developed to inhibit the DNA synthesis activity of the HBV RT. Furthermore, additional activities of the RT that are also essential to HBV replication, including specific recognition of the viral RNA and its packaging into viral nucleocapsids, may be exploited for antiviral development. To achieve a more potent inhibition of viral replication and ultimately cure chronic HBV infection, the next generation of anti-HBV therapies will likely need to include NRTIs, NNRTIs, and other agents that target the viral RT as well as other viral and host factors in various combinations. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B."
    Antiviral research 09/2015; 123. DOI:10.1016/j.antiviral.2015.09.011 · 3.94 Impact Factor
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    • "The first category consists of the nucleos(t)ide RT inhibitors (NRTIs), which are analogs of the natural nucleosides. Most NRTIs lack a 3’-OH and act as chain terminators by blocking DNA polymerization [1-8]. The other group includes the nonnucleoside RT inhibitors (NNRTIs), which are non-competitive RT inhibitors with respect to either dNTP or nucleic acid substrates and block DNA synthesis by binding to a hydrophobic pocket of RT [9-15]. "
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    ABSTRACT: The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF).4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials. Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3' primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent. We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.
    Retrovirology 06/2013; 10(1):65. DOI:10.1186/1742-4690-10-65 · 4.19 Impact Factor
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    • "As ploymerization proceeds, RNase H activity degrades RNA of the RNA/DNA replication intermediates [21]. Although RNase H activity and its interplay with polymerase activity have well-documented [22] [23], it remains unclear how RT–RNase H distinguishes between RNA/DNA hybrid and the structurally similar RNA/RNA homoduplex. For model simplification and specific functional characterizations of IN–RT interactions, we have not involved the documented interplay between RT-polymerase and RNase H activities. "
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    ABSTRACT: Human immunodeficiency virus (HIV) infection yields a high level of non-integrated viral DNA in the infected cells and up to 99% of total viral DNA can be capable of transcription. This capability of non-integrated viral DNA is reducing the efficacy of anti-HIV drug development approaches that solely focus on the integration reactions of viral replication. Using kinetic modeling, we show the transient coordinated regulation of viral DNA production by retrovirus-encoded interacting enzymes reverse transcriptase (RT) and integrase (IN), and thus we identify a possible mechanism for reducing overall efficiency of viral replication. The results indicate that both IN.RT and IN.DNA complexes and their formation rates affect RT processivity and thereby the viral DNA expression level within the pre-integration complex.
    FEBS letters 12/2012; 587(5). DOI:10.1016/j.febslet.2012.12.007 · 3.17 Impact Factor
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