Drug Resistant HIV Reverse Transcriptase

DESCRIPTION (provided by the applicant): HIV-1 RT continues to be a key target for anti-HIV therapy. The long term goal of our project is to comprehensively define HIV RT function and the mechanism of drug resistance. There is an increasing realization that proper positioning of template-primer is central to several key aspects of RT function. We intend to delineate the functional role of three specific interactions between HIV-1 RT and the template-primer. These contact points lie along the template-primer cleft, in front of the active site (contacting the template 5'-overhang), behind the active site (contacting the template at penultimate basepair), or further away in the minor groove (contacting the primer at 3rd to 6th basepairs) respectively. Three specific aims are listed. First, we will study the fingers subdomain-template contacts near the active site via nested deletions of 33-[M loop of HIV-1 RT created to disrupt the proposed interactions with the template overhang and/or the dNTP. In addition to testing these mutants for dNTP-binding, accuracy of dNTP insertion, processivity and strand transfer, we will also test the role of J33-j34 loop as a determinant of the natural high susceptibility of HIV-1 RT to 2',3'-dideoxyNTPs. The mutation rate of multi-dideoxynucleoside analog resistant RT variants and whether a proofreading-like activity contributes to the overall fidelity will be tested. Second, we will investigate the role of the template grip 135a, which forms a close contact with the penultimate basepair of the template-primer, in RT function. We will both attempt to delineate the mechanism of frameshift mutagenesis by HIV-1 RT, the precise role of template grip 135a in frameshift fidelity and the mechanistic basis for its global influence on fidelity. Altered template grip mutants of HIV-1 RT will be tested for effects on affinity to the template-primer duplex, processivity, RNase H activity and strand transfer. Lastly, we will test whether the thumb helix clamp involving aH and cxl helices indeed serves a critical function in enzyme translocation subsequent to each dNTP addition cycle. We will also test its role in processive polymerization and in frameshift fidelity.