Mechanism and fidelity of HIV reverse transcriptase.

Kati, Warren M.; Johnson, Kenneth A.; Jerva, L. F.; Anderson, Karen S.

doi:10.1016/s0021-9258(18)35706-5

Cited by 508 publications

(388 citation statements)

References 44 publications

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“…Very limited data exists on mutation frequencies associated with viral RNA dependent RNA polymerases (RdRps). However, biochemical assays of misincorporation kinetics on RNA templates by the poliovirus RdRp [ 32 , 33 ] or retroviral reverse transcriptase [ 34 ] did not identify C->U misincorporation to be favoured over other mutations.…”

Section: Discussionmentioning

confidence: 99%

Extensive C->U transition biases in the genomes of a wide range of mammalian RNA viruses; potential associations with transcriptional mutations, damage- or host-mediated editing of viral RNA

Simmonds

Ansari

2021

PLoS Pathog

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The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x–7.5x), with a consistently observed favoured 5’ U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11–14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.

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Section: Discussionmentioning

confidence: 99%

Extensive C->U transition biases in the genomes of a wide range of mammalian RNA viruses; potential associations with transcriptional mutations, damage- or host-mediated editing of viral RNA

Simmonds

Ansari

2021

PLoS Pathog

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“…The fidelity mechanisms by which DNA polymerases select the correct, but not incorrect, nucleotide require further elucidation at the level of atomic resolution. On the basis of extensive kinetic measurements and a large body of crystallographic structures for several DNA polymerases (e.g., Escherichia coli DNA polymerase I Klenow fragment (Kuchta et al, 1988;Dahlberg and Benkovic, 1991;Ollis et al, 1985;Beese et al, 1993), phage T7 DNA polymerase (Patel et al, 1991;Doublié et al, , 1999, HIV-1 reverse transcriptase (Kati et al, 1992;Huang et al, 1998;Ding et al, 1998), phage T4 DNA polymerase (Frey et al, 1995), and DNA polymerase b (Beard et al, 2002a;Beard and Wilson, 2003;Vande Berg et al, 2001;Zhong et al, 1997Zhong et al, , 1998Kraynov et al, 1997;Ahn et al, 1997Ahn et al, , 1998Werneburg et al, 1996;Sawaya et al, 1997Sawaya et al, , 1994Pelletier et al, 1994)), the overall pathway of incorporation of a correct 2#-deoxyribonucleoside 5#-triphosphate (dNTP) by a DNA polymerase has been proposed, as sketched in Fig. 1.…”

Section: Introductionmentioning

confidence: 99%

Highly Organized but Pliant Active Site of DNA Polymerase β: Compensatory Mechanisms in Mutant Enzymes Revealed by Dynamics Simulations and Energy Analyses

Yang

Beard

Wilson

et al. 2004

Biophysical Journal

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To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg(258) is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe(272), Asn(279), and Arg(283)) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg(258)) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.

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“…Some enzymes were further purified by heparin-Sepharose (GE Healthcare) affinity chromatography, as previously described [40]. RTs were quantified by active site titration before biochemical studies, using template-primer D38/25PGA [38,41].…”

Section: Expression and Purification Of Recombinant Rtsmentioning

confidence: 99%

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction

Saura¹,

Sapena-Ventura²,

Luczkowiak³

et al. 2021

Viruses

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HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342–351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of β-sheets 17 and 18 and their connecting loop (residues 342–350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.

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Mechanism and fidelity of HIV reverse transcriptase.

Cited by 508 publications

References 44 publications

Extensive C->U transition biases in the genomes of a wide range of mammalian RNA viruses; potential associations with transcriptional mutations, damage- or host-mediated editing of viral RNA

Extensive C->U transition biases in the genomes of a wide range of mammalian RNA viruses; potential associations with transcriptional mutations, damage- or host-mediated editing of viral RNA

Highly Organized but Pliant Active Site of DNA Polymerase β: Compensatory Mechanisms in Mutant Enzymes Revealed by Dynamics Simulations and Energy Analyses

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction

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