Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg(2+), Mn(2+), Co(2+) or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn(2+) and Co(2+) compared with Mg(2+). Activation of RNase H by these metal ions followed sigmoidal dose–response curves suggesting cooperative metal ion binding. Titration of Mg(2+)-bound HIV RNase H with Mn(2+) or Co(2+) ions generated bell-shaped activity dose–response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn(2+) and Co(2+) concentrations, and complete replacement of Mg(2+) occurred at higher Mn(2+) or Co(2+) concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg(2+), is significantly inhibited by submillimolar concentrations of Mn(2+) and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H