of Work: The human immunodeficiency virus (HIV-1) has high mutation rates within certain portions of its genome, permitting rapid evolution of new forms of the the virus that are resistant to drug treatments or can evade the hosts immune response. This project investigates the most likely cause for this, inaccurate DNA synthesis by the HIV-1 reverse transcriptase (RT). Most mistakes made in vitro are initiated by slippage of the template and primer strands, perhaps when the polymerase dissociates and then reassociates with the DNA. In order to gain insight into why the HIV-1 RT is so error-prone for these types of mistakes, we are examining mutant derivatives of HIV-1 RT, with emphasis on amino acids believed to be important for template- primer interactions. We previously identified five amino acids that comprise a minor groove binding track (MGBT) that contacts the template-primer just back from the active site. This year, we completed the analysis of seven mutants with different amino acids substituted for the most critical MGBT residue, W266. The results suggest that hydrophobic interactions make a major contribution to the stability of the RToDNA complex. This may be a general feature of lentiviral RTs that is distinctive in comparison to how normal cellular DNA polymerases bind to template-primers. We are continuing to examine a number of mutants with amino acid changes nearer to the polymerase active site, in the primer grip and fingers subdomain. Analyses of this type enhance our understanding of how RT interact with substrates and may provide insights into the hypermutability of the AIDS virus. Since many of these studies focus on mutations resulting from strand slippage, they may also provide insights relevant to several human diseases characterized by mutations that may result from strand slippage during DNA replication. - Aids, DNA Polymerase, Hypermutations, Mutagenesis, Nucleoside Inhibitors, Replication Fidelity, Transcriptase
