18th International HIV Drug Resistance Workshop Basic Principles & Clinical Implications June 9–13 2009, Fort Myers, Florida, USA

MUTATIONAL ANALYSIS OF RESIDUE ASN348 IN HIV-1 REVERSE TRANSCRIPTASE

Antivir Ther 2009; 14 Suppl 1:A32 (abstract no. 30)

J Radzio¹, G Tachedjian² and N Sluis-Cremer^1
1University of Pittsburgh, Division of Infectious Diseases, Pittsburgh, PA, USA; ²Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Australia

BACKGROUND: We previously demonstrated that the N348I mutation in the connection domain of HIV-1 reverse transcriptase (RT) confers both zidovudine (AZT) and nevirapine resistance. However, residue N348 in both subunits of HIV-1 RT resides distal to the DNA polymerase active site, the RNase H active site, the template/ primer (T/P) binding tract and the NNRTI-binding pocket. Accordingly, the structural mechanism by which N348I confers dual AZT/nevirapine resistance is not known. In the current study, we carried out mutational analyses of residue N348 to specifically address structure-activity resistance relationships.

METHODS: The N348A, N348E, N348I, N348L, N348Q and N348R mutations were introduced into wild-type (WT) HIV-1 RT and the recombinant enzymes were purified to homogeneity. The DNA polymerase and RNase H activities of the enzymes, as well as their sensitivities to AZT and nevirapine, were determined using appropriate biochemical assays. Molecular modeling analyses using a novel model of HIV-1 RT in complex with an RNA/ DNA T/P were performed to complement the biochemical analyses.

RESULTS: The N348A, N348I, N348L and N348Q enzymes exhibited comparable DNA polymerase activities to WT HIV-1 RT. By contrast, the N348E and N348R enzymes exhibited significantly diminished DNA polymerase activities and, accordingly, were excluded from subsequent analyses. ATP-mediated excision assays demonstrated that the efficiency of AZT-monophosphate (MP) was N348I RT>N348L RT>N348Q RT>WT RT≈N348A RT. As expected, the relative AZT-MP excision activity for each enzyme correlated with its RNase H activity. Modeling studies suggest that the N348I, N348L and N348Q mutations perturb the positioning of the β12-β13 loop in the p51 subunit of RT, which directly contacts the RNA strand of an RNA/DNA T/P, thus providing a plausible structural mechanism for AZT resistance. However, the N348A, N348I, N348L and N348Q enzymes all demonstrated similar levels of nevirapine resistance and there was no correlation between nevirapine resistance and RNase H activity.

CONCLUSIONS: These analyses further support previous findings that N348I confers AZT resistance via an RNase-H-dependent mechanism. However, nevirapine resistance appears to be independent of the RNase H activity of the enzyme. Additional mechanistic studies, including subunit selective mutational analyses, are currently in progress.

2009-06-09
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