Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural RNA.
The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the phi Xam 16 reversion assay. A phi X174 sequence harboring the amber 16 codon was cloned into a transcription vector. RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT. An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription. The frequency of dGTP.U mismatches was determined to be 1/360, while dGTP.rA mismatches formed at a rate of 1/4600. These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template. Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given. The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.
Universität Bayreuth, Laboratorium für Biochemie, Germany.
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A
Hübner
M
M
Kruhoffer
F
F
Grosse
G
G
Krauss
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic
D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase