p66 Trp24 and Phe61 Are Essential for Accurate Association of HIV-1 Reverse Transcriptase with Primer/Template

Preventing dimerization of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) constitutes an alternative strategy to abolish virus proliferation. We have previously demonstrated that a short peptide derived from the Trp cluster of the connection domain disrupts the RT dimer by interacting with Trp24 and Phe61 in a cleft located between the fingers and the connection domains of p51. Both Trp24 and Phe61 of p51 are essential for the stability of the RT dimer. Here, in order to understand the requirement of Trp24 and Phe61 in the p66 subunit, we have investigated their implication in the formation of RT–primer/template (p/t) complexes and in RT processivity by combining pre-steady-state and steady-state kinetics with site-directed mutagenesis. We demonstrate that both residues are essential for proper binding of the p/t and control conformational changes required for RT ordered mechanism. Trp24 and Phe61 act on p/t binding and remodeling of the catalytic site. Phe61G mutation increases the binding “on” rate of both p/t and mismatched p/t, yielding an unfavorable RT–p/t for polymerase catalysis, unable to pursue mispair extension. Considering the structure of unliganded RT, Phe61 seems to be involved in the dynamics of p66 thumb–finger interactions and in stabilization of the p/t in the catalytic site. In contrast, the p66 Trp24G mutation alters the overall kinetics of p/t binding and is essentially involved in stabilizing the RT–p/t complex by contacting the 5′ overhang of the template strand. Mutation of both Trp24 and Phe61 alters mispair extension efficiency, suggesting that disruption of the tight contacts between the fingers domain and the 5′ overhang of the template strand increases RT fidelity and reduces RT processivity. Taken together, these studies infer that mutations altering the aromatic nature of Phe61 or Trp24 that may occur to counteract peptide inhibitors targeting this region will generate an unstable RT exhibiting low polymerase activity and higher fidelity. As such, our work suggests that the combined application of peptide-based RT dimerization inhibitors is likely to be highly efficient.