Fidelity of HIV-1 reverse transcriptase copying RNA in vitro.
The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
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Loeb
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004279:DNA, Viral / Q000096:biosynthesis; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000737:chemistry*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase