1 research outputs found
Major Groove Binding Track Residues of the Connection Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Enhance cDNA Synthesis at High Temperatures
At
high temperatures, RNA denaturation can improve the efficiency
and specificity of reverse transcription. Refined structures and molecular
models of HIV-1 reverse transcriptases (RTs) from phylogenetically
distant clades (i.e., group M subtype B and group O) revealed a major
interaction between the template-primer and the Arg<sup>358</sup>-Gly<sup>359</sup>-Ala<sup>360</sup> triad in the large subunit of HIV-1<sub>M/B</sub> RT. However, fewer contacts were predicted for the equivalent
Lys<sup>358</sup>-Ala<sup>359</sup>-Ser<sup>360</sup> triad of HIV-1<sub>O</sub> RT and the nucleic acid. An engineered HIV-1<sub>O</sub> K358R/A359G/S360A
RT showed increased cDNA synthesis efficiency above 68 Ā°C, as
determined by qualitative and quantitative reverse transcription polymerase
chain reactions. In comparison with wild-type HIV-1<sub>O</sub> RT,
the mutant enzyme showed higher thermal stability but retained wild-type
RNase H activity. Mutations that increased the accuracy of HIV-1<sub>M/B</sub> RTs were tested in combination with the K358R/A359G/S360A
triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I)
had a negative effect on reverse transcription efficiency above 65
Ā°C. RTs with improved DNA binding affinities also showed higher
cDNA synthesis efficiencies at elevated temperatures. Two of the most
thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q)
showed moderately increased fidelity in forward mutation assays. Our
results demonstrate that the triad of Arg<sup>358</sup>, Gly<sup>359</sup>, and Ala<sup>360</sup> in the major groove binding track of HIV-1
RT is a major target for RT stabilization, and most relevant for improving
reverse transcription efficiency at high temperatures