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 0) revealed a major interaction between the template-primer and the Arg(358)-Gly(359)-Ala(360) triad in the large subunit of HIV-1(M/B) RT. However, fewer contacts were predicted for the equivalent Lys(358)-Ala(359)-Ser(360) triad of HIV-1(O) RT and the nucleic acid. An engineered HIV-1(O) K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 degrees C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1(O) RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1(M/B) 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 degrees 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(358), Gly(359), and Ala(360) 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.