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

John G. Julias, Mary Jane McWilliams, Stefan G. Sarafianos, Edward Arnold, Stephen H. Hughes

Research output: Contribution to journalArticlepeer-review

89 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)9515-9520
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number14
DOIs
StatePublished - Jul 9 2002

All Science Journal Classification (ASJC) codes

  • General

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