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

Nucleic Acids Res. 2003 Dec 1;31(23):6852-9. doi: 10.1093/nar/gkg881.


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.

MeSH terms

  • Binding Sites
  • Cations, Divalent / metabolism*
  • Cations, Divalent / pharmacology
  • Drug Design*
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / enzymology
  • HIV / enzymology*
  • Metals / metabolism*
  • Metals / pharmacology
  • Ribonuclease H / antagonists & inhibitors*
  • Ribonuclease H / metabolism*
  • Substrate Specificity


  • Cations, Divalent
  • Enzyme Inhibitors
  • Metals
  • Ribonuclease H