title: Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis.
abstract: Asp(443) and Glu(478) 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 Asp(443) or Gln for Glu(478) 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.
authors: Centro de Biología Molecular Severo Ochoa Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, 28049 Madrid, Spain.
authors: Mar
authors: M
authors: Álvarez
authors: Verónica
authors: V
authors: Barrioluengo
authors: Raquel N
authors: RN
authors: Afonso-Lehmann
authors: Luis
authors: L
authors: Menéndez-Arias
mesh_terms: D019943:Amino Acid Substitution; D004247:DNA / Q000096:biosynthesis*; D017931:DNA Primers / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017434:Protein Structure, Tertiary; D054309:Ribonuclease H, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics*; D013698:Templates, Genetic
chemical_list: D017931:DNA Primers; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D054309:Ribonuclease H, Human Immunodeficiency Virus