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.
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.
Julias
John G
JG
HIV Drug Resistance Program, National Cancer Institute, National Institutes of Health, P.O. Box B, Frederick, MD 21702-1201, USA.
McWilliams
Mary Jane
MJ
Sarafianos
Stefan G
SG
Arnold
Edward
E
Hughes
Stephen H
SH
D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D016325:HIV Long Terminal Repeat; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
D004279:DNA, Viral; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H