STRUCTURE-FUNCTION STUDIES OF HIV-1 REVERSE TRANSCRIPTASE

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Summary 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 host's 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 completed studies of mutants with alanine substituted for four specific amino acids in alpha helix H of the thumb subdomain and one palm residue. All have reduced DNA binding affinity, resistance to AZTTP, reduced processivity and/or altered framshift fidelity. They also are altered in their ability to interact with styrene oxide N2-guanine adducts, a probe for interactions in the major and minor grooves of DNA. Modeling suggests that these five amino acids comprise a minor groove binding track (MGBT) that contacts the template-primer just back from the active site. This year we began studies of RT mutants with changes of MGBT residues to amino acids other than alanine, to probe the importance of hydrophobic and H-bonding interactions. We are also examining mutants with amino acid changes nearer to the polymerase active site. Analysis of these enzymes will enhance our understanding of how RT interact with substrates and may provide insights into the hypermutability of the AIDS virus. Since 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.