Thermostable HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase

Barrioluengo, Verónica; Saura, María; Barbieri, Daniela; Menéndez‐Arias, Luis

doi:10.1042/bj20101852

Cited by 26 publications

(57 citation statements)

References 46 publications

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“…In vitro and ex vivo experiments have shown that decreased RNase H activity leads to reduced template switching (55–58). In our assays, based on DNA-dependent DNA synthesis, we found that RNase H-deficient mutants can generate large deletions (and insertions), while these types of errors are rare in RTs with wild-type RNase H activity (22,31). Previous studies showed that RNase H minus HIV-1 RT (i.e.…”

Section: Discussionmentioning

confidence: 83%

“…Phage DNA was obtained from all mutant plaques, and the sequence of the reporter gene was determined in all cases. No lac Z mutations were identified after sequencing phage DNA from >20 000 plaques obtained from two to three E. coli electroporations carried out with gapped M13mp2 DNA substrate (31). For each enzyme, mutant plaques were obtained after transfection with the products of four to six gap-filling reactions.…”

Section: Resultsmentioning

confidence: 99%

“…The fidelity of DNA-dependent DNA synthesis was determined using the forward mutation assay (24), in the previously described conditions (31). Briefly, a gapped M13mp2 DNA substrate was obtained by deleting 407 nucleotides of one of the strands, including the lacZ α-complementation target sequence.…”

Section: Methodsmentioning

confidence: 99%

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

Saura

Barrioluengo

Afonso-Lehmann

et al. 2013

Nucleic Acids Research

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Asp443 and Glu478 are essential active site residues in the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We have investigated the effects of substituting Asn for Asp443 or Gln for Glu478 on the fidelity of DNA-dependent DNA synthesis of phylogenetically diverse HIV-1 RTs. In M13mp2 lacZα-based forward mutation assays, HIV-1 group M (BH10) and group O RTs bearing substitutions D443N, E478Q, V75I/D443N or V75I/E478Q showed 2.0- to 6.6-fold increased accuracy in comparison with the corresponding wild-type enzymes. This was a consequence of their lower base substitution error rates. One-nucleotide deletions and insertions represented between 30 and 68% of all errors identified in the mutational spectra of RNase H-deficient HIV-1 group O RTs. In comparison with the wild-type RT, these enzymes showed higher frameshift error rates and higher dissociation rate constants (koff) for DNA/DNA template–primers. The effects on frameshift fidelity were similar to those reported for mutation E89G and suggest that in HIV-1 group O RT, RNase H inactivation could affect template/primer slippage. Our results support a role for the RNase H domain during plus-strand DNA polymerization and suggest that mutations affecting RNase H function could also contribute to retrovirus variability during the later steps of reverse transcription.

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Section: Discussionmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

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

Saura

Barrioluengo

Afonso-Lehmann

et al. 2013

Nucleic Acids Research

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“…Interestingly, these mutations were developed by random mutagenesis, but the multiple variant E69K/E302R/ W313F/L435G/N454K has lower K m value for T/P than the wild-type MMLV RT (Aresi and Hogrefe 2009). In HIV-1 RT, it was recently reported that the HIV-1 group O RT is more stable than HIV-1 group M (major-typed) RT and is as stable as MMLV RT (Á lvarez et al 2009;Barrioluengo et al 2011). The sequence homology between these two HIV-1 RTs is 21 %, and the amino acid residues essential for the difference in stability have not been identified.…”

Section: Increase In Rt Stability By Introducing Positive Charges By mentioning

confidence: 99%

Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis

2012

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Avian myeloblastosis virus reverse transcriptase (AMV RT) is a heterodimer consisting of a 63 kDa α-subunit and a 95 kDa β subunit. Moloney murine leukaemia virus reverse transcriptase (MMLV RT) is a 75 kDa monomer. These two RTs are the most extensively used for conversion of RNA to DNA. We previously developed several mutations that increase the thermostability of MMLV RT and generated a highly stable MMLV RT variant E286R/E302K/L435R/D524A by combining three of them (Glu286→Arg, Glu302→Lys, and Leu435→Arg) and the mutation to abolish RNase H activity (Asp524→Ala) [Yasukawa et al. (2010) J Biotechnol 150:299-306]. To generate a highly stable AMV RT variant, we have introduced the triple mutation of Val238→Arg, Leu388→Arg, and Asp450→Ala into AMV RT α-subunit and the resulted variant V238R/L388R/D450A, was expressed in insect cells and purified. The temperature decreasing the initial activity by 50 %, measured over 10 min, of the variant with or without template primer (T/P), poly(rA)-p(dT)(15), was 50 °C; for the wild-type AMV RT α-subunit (WT) this was 44 °C. The highest temperature at which the variant exhibited cDNA synthesis activity was 64 °C; the WT was 60 °C. A highly stable AMV RT α-subunit is therefore generated by the same mutation strategy as applied to MMLV RT and that positive charges are introduced into RT at positions that have been implicated to interact with T/P by site-directed mutagenesis.

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“…However, without a precise comparison of fidelity and processivity, it is difficult to determine which variant is the most suitable. To address this, further characterization of the thermostable variants generated previously (MM3 and MM4) 8) and in this study (MM7 and MM8) by misincoporation, mispair extension, and primer extension assay 17,18) is currently in progress.…”

mentioning

confidence: 99%

Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

Konishi

Yasukawa

2014

Bioscience, Biotechnology, and Biochemistry

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After thermal incubation at 48 °C for 10 min, single variants of Moloney murine leukemia virus reverse transcriptase, V433R and V433K in which a surface hydrophobic residue, Val433, was mutated, retained 55% of initial reverse transcription activity, while the wild-type enzyme retained 17%. After thermal incubation at 50 °C for 10 min, multiple variants D108R/E286R/V433R and D108R/E286R/V433R/D524A, in which Val433→Arg was combined with stabilizing mutations we identified previously, Asp108→Arg and Glu286→Arg, and RNase H activity-eliminating mutation Asp524→Ala, retained 70% of initial activity, exhibiting higher stability than V433R or V433K.

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Thermostable HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase

Cited by 26 publications

References 46 publications

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

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

Improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis

Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

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