Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

Daddacha, Waaqo; Noble, Erin; Nguyen, Laura A.; Kennedy, Edward M.; Kim, Baek

doi:10.1074/jbc.m113.458398

Cited by 7 publications

(16 citation statements)

References 50 publications

(69 reference statements)

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“…An embedded rNMP in a DNA template increases the pausing of cellular DNA polymerases, which is associated with increased mutation during DNA synthesis (17,18). Organisms commonly harbor repair mechanisms that specifically remove rNMPs incorporated in dsDNA, most likely to avoid genomic mutations (13,16,19,20).…”

Section: Introductionmentioning

confidence: 99%

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Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template

et al. 2015

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Non-dividing macrophages maintain extremely low cellular deoxyribonucleotide triphosphate (dNTP) levels, but high ribonucleotide triphosphate (rNTP) concentrations. The disparate nucleotide pools kinetically forces Human Immunodeficiency Virus 1 (HIV-1) reverse transcriptase (RT) to incorporate non-canonical rNTPs during reverse transcription. HIV-1 RT pauses near ribonucleoside monophosphates (rNMPs) embedded in the template DNA, which has previously been shown to enhance mismatch extension. Here, pre-steady state kinetic analysis shows rNTP binding affinity (Kd) of HIV-1 RT for non-canonical rNTPs was 1.4- to 43-fold lower, and the rNTP rate of incorporation (kpol) was 15- to 1551-fold slower than for dNTPs. This suggests that RT is more selective for incorporation of dNTPs rather than rNTPs. HIV-1 RT selectivity for dNTP versus rNTP is the lowest for ATP, implying that HIV-1 RT preferentially incorporates ATP when dATP concentration is limited. We observed that incorporation of a dNTP occurring one nucleotide before an embedded rNMP in the template had a 29-fold greater Kd and a 20-fold slower kpol as compared to the same template containing dNMP. This reduced the overall dNTP incorporation efficiency of HIV-1 RT by 581-fold. Finally, the RT mutant Y115F displayed lower discrimination against rNTPs due to its increase in binding affinity for non-canonical rNTPs. Overall, these kinetic results demonstrate that HIV-1 RT utilizes both substrate binding and a conformational change during: 1) enzymatic discrimination of non-canonical rNTPs from dNTPs and 2) during dNTP primer extension with DNA templates containing embedded rNMP.

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Section: Introductionmentioning

confidence: 99%

“…Organisms commonly harbor repair mechanisms that specifically remove rNMPs incorporated in dsDNA, most likely to avoid genomic mutations (13,16,19,20). We recently reported that HIV-1 RT also paused near rNMP sites in the DNA template during DNA synthesis (17). …”

Section: Introductionmentioning

confidence: 99%

Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template

et al. 2015

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“…Our observation of the anti-HIV-1 activity of 2=-C-methylcytidine provides one line of support for the direct incorporation of the ribonucleoside analog, as the 2=-C-methyl group likely prevents reduction to the deoxyribonucleoside form. Incorporation of a ribonucleoside analog not only could reduce viral DNA synthesis but also could increase the viral mutation rate (12).…”

Section: Discussionmentioning

confidence: 99%

“…In HIV-1 RT, Tyr-115 plays a role as a steric gate in preventing the incorporation of nucleotides with a 2=-hydroxyl group in a cation-independent manner, while its influence on fidelity was found to be modulated by Mg 2ϩ or Mn 2ϩ (11). Several observations, including those obtained from monocyte-derived macrophages, have found that ribonucleotides and their analogs can impact reverse transcription (12)(13)(14). Quantita-tive tandem mass spectrometry was used to demonstrate that macrophages have deoxynucleotide triphosphate (dNTP) concentrations 22-to 320-fold lower than those observed in dividing target cells, along with a greater disparity between NTP and dNTP concentrations (14).…”

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Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1

Dapp

Bonnac

Patterson

et al. 2014

J Virol

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Reverse transcription is an important early step in retrovirus replication and is a key point targeted by evolutionarily conserved host restriction factors (e.g., APOBEC3G, SamHD1). Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target of antiretroviral drugs, and concerns regarding drug resistance and off-target effects have led to continued efforts for identifying novel approaches to targeting HIV-1 RT. Several observations, including those obtained from monocytederived macrophages, have argued that ribonucleotides and their analogs can, intriguingly, impact reverse transcription. For example, we have previously demonstrated that 5-azacytidine has its greatest antiviral potency during reverse transcription by enhancement of G-to-C transversion mutations. In the study described here, we investigated a panel of ribonucleoside analogs for their ability to affect HIV-1 replication during the reverse transcription process. We discovered five ribonucleosides-8-azaadenosine, formycin A, 3-deazauridine, 5-fluorocytidine, and 2=-C-methylcytidine-that possess anti-HIV-1 activity, and one of these (i.e., 3-deazauridine) has a primary antiviral mechanism that involves increased HIV-1 mutational loads, while quantitative PCR analysis determined that the others resulted in premature chain termination. Taken together, our findings provide the first demonstration of a series of ribonucleoside analogs that can target HIV-1 reverse transcription with primary antiretroviral mechanisms that include premature termination of viral DNA synthesis or enhanced viral mutagenesis.

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“…The direct incorporation of 5-aza-C as a ribonucleotide (5-aza-CTP) was predicted to lead to a mutational pattern distinct from that of 5-aza-dCTP, because incorporated ribonucleotides can (i) be replaced (correctly or incorrectly) by RNase H2-mediated repair, (ii) cause mutations if left unrepaired, particularly short (2-to 5-bp) deletions, and (iii) elevate the HIV-1 RT mismatch extension frequency on the opposite strand (26)(27)(28). To prepare samples for sequencing, U373-MAGI cells were treated with DMSO (i.e., no drug), 5-aza-C (EC 75 , ϳ260 M) or 5-aza-dC (EC 75 , ϳ3.8 M).…”

Section: -Azacytidine and 5-aza-2=-deoxycytidine Induce Similar Levementioning

confidence: 99%

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

Rawson

Daly

Xie

et al. 2016

Antimicrob Agents Chemother

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h 5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2=-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTP in vitro at least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses. RNA viruses exhibit high mutation rates and have been postulated to replicate near the error threshold-the maximal mutation rate compatible with the maintenance of genetic information (1, 2). Thus, these viruses may be particularly sensitive to small molecules that promote viral mutations, an antiviral strategy called lethal mutagenesis (3). Lethal mutagenesis has been pursued as a potential antiviral approach for many different RNA viruses (4). Most small-molecule candidates for lethal mutagenesis identified thus far have been nucleoside analogs with altered base-pairing properties. These nucleoside analogs base pair promiscuously due to ionization, structural rearrangement, tautomerization, or conformational flexibility (5). Alternatively, small molecules can be used to promote viral mutagenesis by leveraging host nucleic acid-editing enzymes that are part of the innate immune response. For example, molecules have been identified that block the degradation of APOBEC3 enzymes by the human immunodeficiency virus type 1 (HIV-1) accessory protein Vif (6-9). These molecules ultimately promote the incorporation of APOBEC3 proteins into virions, resulting in lethal G-to-A hypermutation during the next cycle of replication.The ribonucleoside analog 5-azacytidine (5-aza-C) reduces the infectivity of HIV-1 by inducing lethal mutagenesis (10). 5-Aza-C is active during both the early and late phases of viral replication, reflecting incorporation during both r...

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Effect of Ribonucleotides Embedded in a DNA Template on HIV-1 Reverse Transcription Kinetics and Fidelity

Cited by 7 publications

References 50 publications

Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template

Pre-steady state kinetic analysis of HIV-1 reverse transcriptase for non-canonical ribonucleoside triphosphate incorporation and DNA synthesis from ribonucleoside-containing DNA template

Discovery of Novel Ribonucleoside Analogs with Activity against Human Immunodeficiency Virus Type 1

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2′-Deoxycytidine

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