A critical feature of the life cycle the human immunodeficiency virus (HIV-1) that causes Acquired Immunodeficiency Syndrome (AIDS) is its ability to gene-rate diversity. HIV-1 has exceptionally high mutation rates within certain portions of its genome, permitting rapid evolution of new forms of the virus that are able to evade the host's immune response. In order to determine if errors committed by the viral reverse transcriptase could account for diversity in vivo, we previously examined the accuracy of HIV-1 reverse transcriptase (RT) using in vitro fidelity assays and found this enzyme to be exceptionally error-prone. Sequence analysis of mutants resulting from in vitro synthesis demonstrates that the enzyme produces base substitution and one-base frameshift mutational hotspots that may result from template-primer slippage. We have obtained strong evidence that the frameshifts are indeed due to misalignment. Using a steady-state enzyme kinetic analysis of an exceptional base-substitution hot spot, we have also established equally strong support for a model for base substitutions generated by transient misalignment. Processivity analysis for the enzyme on the M13mp2 DNA template reveals strong termination at specific sites within homopolymer sequences that correlate with frameshift mutational hot spots. Since these results suggest that the formation and/or utilization of misaligned template-primers is increased during the dissociation-reinitiation phase of the reaction, we have been examining the relationship between termination and error rates in more detail. We have also initiated studies to examine the fidelity of reverse transcriptases during copying of RNA templates and studies of the effects of AZT metabolites on both the HIV-1 reverse transcriptase and human cell replication and mismatch repair. These efforts will continue to focus on elucidating the mechanisms responsible for the error-proness of HIV-1 RT, in the hope that this provide insights into the interaction of the enzyme's active site with its substrates and may be useful in designing RT-targeted drugs.
