Unequal human immunodeficiency virus type 1 reverse transcriptase error rates with RNA and DNA templates.

Abstract

Sequence variation in the type 1 human immunodeficiency virus (HIV-1) results, in part, from inaccurate replication by reverse transcriptase. Although this enzyme is error-prone during synthesis in vitro with DNA templates, the fidelity of RNA-dependent DNA synthesis relevant to minusstrand replication in the virus life cycle has not been examined extensively. In the present study, we have developed a system to determine the fidelity of transcription and reverse transcription and have used it to compare the fidelity of DNA synthesis by the HIV-1 reverse transcriptase with RNA and DNA templates of the same sequence. Overall, fidelity was several-fold higher with RNA than with DNA. Sequence analysis of mutants generated with the two substrates revealed that differences in error rates were substantial for specific errors. Fidelity with RNA was >10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Because such errors likely result from template-primer slippage, this result suggests that misaligned intermediates are formed and/or used less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 reverse transcriptase synthesis with an RNA template-DNA primer was error-prone during incorporation of the first two nucleotides, perhaps due to aberrant enzyme-substrate interactions as synthesis initiates. The unequal error rates with RNA and DNA templates suggest that mistakes during minus-and plus-strand DNA synthesis may not contribute equally to the mutation rate of HIV-1. The data also provide estimates of substitution and frameshift error rates during transcription by T7 RNA polymerase.The causative agent of AIDS, human immunodeficiency virus type 1 (HIV-1), exhibits extensive genomic heterogeneity (1). One source of this sequence diversity is inaccurate DNA replication by its reverse transcriptase (RT). Conversion of the viral genomic RNA to double-stranded DNA is a two-step process. The minus strand is synthesized by using viral RNA as a template. The RT then uses this newly synthesized, complementary DNA as a template for secondstrand synthesis.HIV-1 RT, which lacks a 3'--5' exonuclease proofreading activity (2), has been shown to be error-prone during DNAdependent DNA synthesis in vitro (2-7). Although such studies are relevant to second-strand synthesis, the fidelity of reverse transcription of RNA to DNA is also relevant to retroviral mutagenesis. Limited information is available on the fidelity of RNA-dependent DNA synthesis by HIV-1 RT (8-10). We therefore decided to examine HIV-1 RT fidelity with heteropolymeric RNA by adapting a forward mutation assay (2) previously used to establish HIV-1 RT error rates with a DNA template (4). This assay has permitted a direct comparison of fidelity of HIV-1 RT with RNA and DNA templates of the same sequence as well as an estimate for error rates during transcription by T7 RNA polymerase. MATERIALS AND METHODSEnzymes and Reagents. The recombinant form o...

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Review

“…Furthermore, the mutations appear to be nonrandom and can include substitutions, insertions and deletions (Preston et a/., 1988; Roberts et al, 1988). Fidelity is several fold higher with RNA than with DMA, suggesting that most mutations occur during the second DMA strand synthesis with the DMA template-DNA primer (Boyer, J.C. et al, 1992). Based on the cumulative frequencies of base substitutions, frameshifts and deletions/insertions, about 10 to 100% of replicating viruses will produce mutant progeny after each replication cycle.…”