FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASES

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Sequence variation in the genome of the human immunodeficiency virus (HIV- 1) results from inaccurate replication by reverse transcriptase. This enzyme is error-prone during synthesis in vitro with DNA templates. However, the fidelity of RNA-dependent DNA synthesis relevant to minus- strand replication in the virus life cycle had not been examined previously. This year we developed an assay system to determine the fidelity of transcription and reverse transcription and 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 more than 10-fold higher for substitution and minus-one nucleotide errors at five different homopolymeric positions. Since such errors likely result from template- primer slippage, this result suggests that misaligned intermediates are formed and/or utilized less frequently with an RNA template-DNA primer than with a DNA template-DNA primer. The results also suggest that HIV-1 RT 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. We also completed studies of the effects of AZT metabolites on normal cellular replication and on mismatch repair and studies of the RT from AZT-resistant virus clones. We will continue to focus on elucidating the mechanisms responsible for the error-proneness of HIV-1 RT, in the hope that this will provide insights into the interaction of the enzyme's active site with its substrates that may be useful in designing RT targeted drugs.