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

Abstract

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 Mg²⁺, Mn²⁺, Co²⁺ or a combination of these divalent metal ions. Polymerase‐independent HIV RNase H was similar to or more active with Mn²⁺ and Co²⁺ compared with Mg²⁺. Activation of RNase H by these metal ions followed sigmoidal dose–response curves suggesting cooperative metal ion binding. Titration of Mg²⁺‐bound HIV RNase H with Mn²⁺ or Co²⁺ 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 Mn²⁺ and Co²⁺ concentrations, and complete replacement of Mg²⁺ occurred at higher Mn²⁺ or Co²⁺ 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 Mg²⁺, is significantly inhibited by submillimolar concentrations of Mn²⁺ 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.