Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

Ehteshami, Maryam; Scarth, Brian J.; Tchesnokov, Egor P.; Dash, Chandravanu; Grice, Stuart F. J. Le; Hallenberger, Sabine; Jochmans, Dirk; Götte, Matthias

doi:10.1074/jbc.m804882200

Cited by 46 publications

(63 citation statements)

References 29 publications

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“…The prototype compound, INDOPY-1 (which is active against NNRTI-resistant HIV), binds and stabilizes RT-DNA/DNA complexes, trapping them in a posttranslocational state. INDOPY-1 binding depends on the chemical nature of the ultimate base pair at the primer 3Ј terminus rather than the chemical nature of the templated base engaged in classic base pairing (71,72). Although the clinical benefits of RT inhibitors in reducing rates of HIV transmission are clear, the rapid emergence of drug resistance continues to pose a challenge (73).…”

Section: Dna Polymerase and Rnase H Inhibitor Developmentmentioning

confidence: 99%

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Grice

2012

Journal of Biological Chemistry

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Synthesis of integration-competent, double-stranded DNA from the (؉)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA-and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (؊)-and (؉)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here. HIV-1 DNA SynthesisThe individual steps of HIV-1 DNA synthesis, catalyzed by the multifunctional reverse transcriptase (RT), 2 are summarized schematically in Fig. 1. (Ϫ)-Strand DNA synthesis, initiated from a cellular tRNA (tRNA 3 Lys ) hybridized to the genomeencoded primer-binding site, continues to the 5Ј terminus, creating (Ϫ)-strong-stop DNA. RNase H-mediated degradation of the resulting RNA/DNA hybrid promotes relocation of nascent (Ϫ)-DNA to the genome 3Ј terminus by a strand transfer event that exploits sequence homology between the 5Ј and 3Ј termini. RNA-templated DNA synthesis continues, accompanied by RNase H-mediated degradation of the RNA genome, the exception to which are two short purine-rich segments (the 3Ј-and central polypurine tracts (PPTs)) from which (ϩ)-strand DNA-dependent DNA synthesis is initiated. Newly synthesized (Ϫ)-strand DNA and 18 nucleotides of the covalently attached tRNA 3 Lys primer provide the template for 3Ј-PPTprimed (ϩ)-strand DNA synthesis until the replication complex stalls at a position corresponding to the first modified tRNA base (A57). As a consequence, the C-terminal RNase H domain is positioned at the (Ϫ)-DNA/tRNA junction, and degradation of the tRNA "template" promotes a second or (ϩ)-strand transfer event supported by homology between (Ϫ)-and (ϩ)-strand DNA primer-binding sites. Although bidirectional DNA synthesis would be sufficient to complete DNA synthesis, HIV utilizes a second, central PPT primer, thereby producing a (ϩ)-strand discontinuity (1). Following (ϩ)-strand transfer, 3Ј-PPT-mediated DNA synthesis continues, displacing ϳ100 nucleotides of central PPT-primed (ϩ)-DNA, but abruptly ceases at the central termination sequence, a prominent feature of which is phased A-tracts that induce minor groove compression (1-3), creating the "central flap" (Fig. 1) (4 -7). Although central flap function remains controversial (8, 9), its mutation or deletion has been shown to impair...

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Section: Dna Polymerase and Rnase H Inhibitor Developmentmentioning

confidence: 99%

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Grice

2012

Journal of Biological Chemistry

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“…These defects result in impaired fitness in vivo, and the M184I mutation is rapidly replaced with M184V during prolonged therapy (52,53). M184V RT also has a mild defect in processivity (3-5, 8, 23, 39, 54) and ternary complex (RT-dNTP-P/T) stability (18,24) accompanied by modest reductions in affinity for natural dNTPs (15,20,60), but this mutant is selected because of a fitness advantage in vivo. Selection experiments in a TAM background resulted in M184V with no evidence of an M184I intermediate (30), and M184V is usually the only mutation detected at RT position 184 in HIV-1-infected patients undergoing prolonged 3TC therapy (53).…”

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A Role of Template Cleavage in Reduced Excision of Chain-Terminating Nucleotides by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing the M184V Mutation

Acosta-Hoyos

Matsuura

Meyer

et al. 2012

J Virol

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Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3′-deoxy-3′-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position −7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the −7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the −7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.

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“…In contrast, the more recently discovered scaffold of indolopyridones (INDOPY-1) (15,16) traps RT in the post-translocational state (15). Owing to its proposed binding mechanism, INDOPY-1 has been referred to as a nucleotide-competing RT inhibit (17).…”

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Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

Auger

Beilhartz

Zhu

et al. 2011

Journal of Biological Chemistry

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The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre-and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA⅐DNA primer/template substrates versus DNA⅐DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA⅐DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.HIV RT is a multifunctional enzyme that catalyzes the conversion of the single-stranded RNA HIV genome to doublestranded DNA that is then incorporated into the host genome by the HIV integrase. In the process, RT must support RNAdirected and DNA-directed DNA synthesis along with ribonuclease H (RNase H) 2 activities to create an integration-competent product. A key step during reverse transcription is the incomplete hydrolysis of the (ϩ)-stranded RNA that leaves behind a purine-rich RNA sequence referred to as the polypurine tract (PPT). The remnant RNA PPT sequence serves as a primer to initiate synthesis of the (ϩ)-strand DNA (second strand) (1-3). The unique structure of the RNA PPT sequence has been shown to be a key determinant of the RNase H cleavage specificity at the PPT/U3 junction (4). A recent report described the mechanism by which RT discriminates between polymerase and RNase H activities thereby enabling more efficient initiation of polymerization from the RNA PPT primer versus other remnant RNA primers (5). Using single-molecule spectroscopy experiments, it was shown that RT binds nucleic acid substrates in two distinct orientations in a manner that is governed by the sugar backbone composition of the four or five nucleotides at each end of the primer. Depending on the binding orientation, RT either initiates polymerization at the 3Ј-end of the primer (polymerase binding mode on a DNA primer), or alternatively, RNA hydrolysis through the RNase H domain (RNase H bindi...

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Mutations M184V and Y115F in HIV-1 Reverse Transcriptase Discriminate against “Nucleotide-competing Reverse Transcriptase Inhibitors”

Cited by 46 publications

References 29 publications

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

A Role of Template Cleavage in Reduced Excision of Chain-Terminating Nucleotides by Human Immunodeficiency Virus Type 1 Reverse Transcriptase Containing the M184V Mutation

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

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