title: Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.
abstract: 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 Arg³⁵⁸-Gly³⁵⁹-Ala³⁶⁰ triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³⁵⁸-Ala³⁵⁹-Ser³⁶⁰ triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/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 °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 Arg³⁵⁸, Gly³⁵⁹, and Ala³⁶⁰ 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.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid , 28049 Madrid, Spain.
authors.forename: Tania
authors.initials: T
authors.lastname: Matamoros
authors.forename: Verónica
authors.initials: V
authors.lastname: Barrioluengo
authors.forename: David
authors.initials: D
authors.lastname: Abia
authors.forename: Luis
authors.initials: L
authors.lastname: Menéndez-Arias
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018076:DNA, Complementary / Q000096:biosynthesis*; D030562:Databases, Protein; D004795:Enzyme Stability; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D050505:Mutant Proteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D013942:Thymine Nucleotides; D004247:DNA; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus; C024157:thymidine 5'-triphosphate