1. Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo.
- Author
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Julias JG, McWilliams MJ, Sarafianos SG, Arnold E, and Hughes SH
- Subjects
- Base Sequence, Cell Line, DNA, Viral genetics, HIV Long Terminal Repeat, HIV Reverse Transcriptase metabolism, Humans, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Structure, Tertiary, Ribonuclease H metabolism, Substrate Specificity, Transcription, Genetic, DNA, Viral biosynthesis, HIV Reverse Transcriptase chemistry, HIV Reverse Transcriptase genetics, HIV-1 genetics, HIV-1 metabolism, Mutation, Ribonuclease H chemistry, Ribonuclease H genetics
- Abstract
Retroviral reverse transcriptases contain a DNA polymerase activity that can copy an RNA or DNA template and an RNase H activity that degrades the viral RNA genome during reverse transcription. RNase H makes both specific and nonspecific cleavages; specific cleavages are used to generate and remove the polypurine tract primer used for plus-strand DNA synthesis and to remove the tRNA primer used for minus-strand DNA synthesis. We generated mutations in an HIV-1-based vector to change amino acids in the RNase H domain that contact either the RNA and DNA strands. Some of these mutations affected the initiation of DNA synthesis, demonstrating an interdependence of the polymerase and RNase H activities of HIV-1 reverse transcription during viral DNA synthesis. The ends of the linear DNA form of the HIV-1 genome are defined by the specific RNase H cleavages that remove the plus- and minus-strand primers; these ends can be joined to form two-long-terminal repeat circles. Analysis of two-long-terminal repeat circle junctions showed that mutations in the RNase H domain affect the specificity of RNase H cleavage.
- Published
- 2002
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