Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis

Saura, María; Barrioluengo, Verónica; Afonso-Lehmann, Raquel N.; Menéndez‐Arias, Luis

doi:10.1093/nar/gkt109

Cited by 21 publications

(18 citation statements)

References 60 publications

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“…These differences are similar to those reported for other primate lentiviral RTs when compared with HIV-1 group M subtype B polymerases, such as the RTs of HIV-1 strains BH10 or HXB2, using the M13-based forward mutation assay. Thus, the HIV-1 BH10 RT showed about two-fold decreased fidelity in comparison with a prototypic WT HIV-1 group O RT5455, while SIV mne and SIV agm RTs were only 1.3 and 1.8 times more accurate, respectively, than HIV-1 group M subtype B RTs5657. Despite the relatively small differences in mutation rates, HIV-1 group M subtype B RTs emerge as the less faithful polymerases among those of primate lentiviruses.…”

Section: Discussionmentioning

confidence: 94%

Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

Saura

Sebastián-Martín

García‐Marquina

et al. 2017

Sci Rep

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Nucleoside reverse transcriptase (RT) inhibitors constitute the backbone of current therapies against human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2, respectively). However, mutational pathways leading to the development of nucleoside analogue resistance are different in both types of HIV. In HIV-2, resistance to all approved nucleoside analogues is conferred by the combination of RT substitutions K65R, Q151M and M184V. Nucleotide incorporation kinetic analyses of mutant and wild-type (WT) HIV-2 RTs show that the triple-mutant has decreased catalytic efficiency due to the presence of M184V. Although similar effects were previously reported for equivalent mutations in HIV-1 RT, the HIV-2 enzymes were catalytically less efficient. Interestingly, in highly divergent HIV-1 RTs, K65R confers several-fold increased accuracy of DNA synthesis. We have determined the intrinsic fidelity of DNA synthesis of WT HIV-2 RT and mutants K65R and K65R/Q151M/M184V. Our results show that those changes in HIV-2 RT have a relatively small impact on nucleotide selectivity. Furthermore, we found that there were less than two-fold differences in error rates obtained with forward mutation assays using mutant and WT HIV-2 RTs. A different conformation of the β3-β4 hairpin loop in HIV-1 and HIV-2 RTs could probably explain the differential effects of K65R.

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Section: Discussionmentioning

confidence: 94%

Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

Saura

Sebastián-Martín

García‐Marquina

et al. 2017

Sci Rep

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“…Glu478→Gln mutations increased the fidelity of HIV-1 O RT(Barrioluengo et al 2011;Álvarez et al 2013). Taken together, these data suggest that the use of multiple HIV-1 O RT variants designed to have even higher stability and fidelity might be the most attractive for practical use in cDNA synthesis in the future.heterodimer.…”

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confidence: 82%

Stabilization of human immunodeficiency virus type 1 reverse transcriptase by site-directed mutagenesis

et al. 2013

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Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer containing 66 kDa p66 and 51 kDa p51 subunits. We previously showed that HIV-1 group M (HIV-1 M) RT and HIV-1 group O (HIV-1 O) RT have higher affinities for dTTP and template-primer (T/P) than Moloney murine leukemia virus RT, which is currently used for cDNA synthesis, suggesting that they might also be useful for cDNA synthesis (Konishi et al. Appl Biochem Biotechnol 2013, 169:77-87). Here, we have increased the thermostability of both HIV-1 M RT and HIV-1 O RT by site-directed mutagenesis. The Asp443 → Ala mutation, which abolishes RNase H activity, was introduced into the p66 subunits of HIV-1 M RT and HIV-1 O RT. The temperatures that reduced the initial activity by 50 % of the resulting mutants, HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51, were 44 and 52 °C, respectively, which were higher than those of wild-type HIV-1 M p66/p51 (42 °C) and HIV-1 O p66/p51 (48 °C). The highest temperature at which both HIV-1 M p66D443A/p51 and HIV-1 O p66D443A/p51 exhibited cDNA synthesis activity was 68 °C, which was higher than for the wild-type enzymes (62 and 66 °C, respectively).

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“…HIV‐1 RT) in DNA‐dependent DNA polymerization assays . Using the forward mutation assay based on the expression of lacZ , error rates of 1.4 × 10 −4 , 5.8 × 10 −5 and 1.2 × 10 −5 have been reported for wild‐type HIV‐1 M/B , HIV‐1 O and MLV RTs, respectively . On the other hand, thermostable MLV RT variants such as SuperScript II and III showed error rates of 3.1 × 10 −5 –6.5 × 10 −5 , which are higher than those obtained with the wild‐type enzyme .…”

Section: Introductionmentioning

confidence: 89%

“…The fidelity of DNA‐dependent DNA synthesis was determined at different temperatures using the M13mp2‐based forward mutation assay , as previously described . Thus, gap filling synthesis reactions were carried out in 25 m m Tris‐HCl (pH 8.0) buffer containing 100 m m KCl, 2 m m DTT, 4 m m MgCl 2 , 250 μ m of each dNTP, 5 μg·mL −1 gapped duplex DNA and 100 n m RT.…”

Section: Methodsmentioning

confidence: 99%

Temperature effects on the fidelity of a thermostable HIV‐1 reverse transcriptase

Saura

Menéndez‐Arias

2013

The FEBS Journal

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Transcriptomics and gene expression analysis are largely dependent of the availability of efficient thermostable reverse transcriptases (RTs). However, the intrinsic fidelity of DNA synthesis catalyzed by retroviral RTs is low. Reported error rates are in the range 1.2 × 10−5–6.7 × 10−4, with oncoretroviral RTs being the most faithful enzymes. Wild‐type HIV‐1 group O (HIV‐1O) RT is a thermostable polymerase that is able to synthesize cDNA at temperatures as high as 70 °C. At 37 °C, its error rate has been estimated at 5.8 × 10−5 in M13mp2 lacZ‐based forward mutation assays. However, at higher temperatures (e.g. 50 and 55 °C), the accuracy of HIV‐1O RT is increased by approximately two‐ to five‐fold. At 55 °C, the HIV‐1O RT error rate (1.3 × 10−5) was similar to that shown by the AffinityScript (Agilent Technologies Inc., La Jolla, CA, USA) RT, a commercially available thermostable murine leukaemia virus RT. At higher temperatures, the increased accuracy of the HIV‐1 enzyme results from a lower base substitution error rate, although it shows a higher tendency to introduce frameshifts. Kinetic studies carried out with model template‐primers suggest minor differences in nucleotide discrimination, although, at higher temperatures, HIV‐1O RT showed a reduced ability to extend mispaired template‐primers.

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Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis

Cited by 21 publications

References 60 publications

Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

Fidelity of classwide-resistant HIV-2 reverse transcriptase and differential contribution of K65R to the accuracy of HIV-1 and HIV-2 reverse transcriptases

Stabilization of human immunodeficiency virus type 1 reverse transcriptase by site-directed mutagenesis

Temperature effects on the fidelity of a thermostable HIV‐1 reverse transcriptase

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