ABSTRACT: Purpose: To determine the biochemical effects of the NNRTI resistance mutations P236L, K103N and Y181C on HIV reverse transcriptase (RT) function.
Methods: Recombinant heterodimeric RTs, consisting of a pNL4-3 backbone with either a P236L, K103N, or Y181C mutation, were expressed in Escherichia coli and purified using metal affinity chromatography. Biochemical properties measured included specific activity of RNA-dependent DNA polymerization on homo- and heteropolymetric templates, processivity of RNA-dependent DNA polymerization on heteropolymeric templates, and the kinetics of formation of RNase H cleavage products. Both modes of RNase H cleavage were assessed. DNA 3'-end-directed RNase H cleavage, which is thought to occur during polymerization, was measured by incubating RT with a template consisting of a 5'-end labeled RNA annealed to DNA such that the DNA 3' end was recessed. RNA 5'-end-directed cleavage, which is essential for the first strong stop strand transfer, was measured using a labeled RNA-DNA hybrid in which the 5' end of the RNA was recessed. Reactions were carried out in the absence of dNTPs. Cleavage products were resolved on denaturing PAGE and quantified using phosphorimaging.
Results: There were no significant difference among the wild-type and three mutant RTs in polymerization specific activities or processivity of DNA polymerization. Both P236L and K103N RTs demonstrated significant slowing of DNA 3'-end directed RNase H cleavage, whereas only P236L demonstrated significant slowing of RNA 5'-end directed RNase H cleavage. Y181C demonstrated a significant acceleration of both DNA 3'-end-directed and RNA 5'-end-directed modes of RNase H cleavage relative to wild-type RT. No aberrantly sized cleavage products were detected with any of the mutant RTs tested.
Conclusions: Some NNRTI-resistant HIV-1 RTs demonstrate abnormalities in RNase H cleavage. Studies to examine additional mutant enzymes are in progress.
