title: Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate.
abstract: The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.
authors.affiliation: Department of Medicine, University of Miami School of Medicine, Florida 33101.
authors.forename: C K
authors.initials: CK
authors.lastname: Tan
authors.forename: R
authors.initials: R
authors.lastname: Civil
authors.forename: A M
authors.initials: AM
authors.lastname: Mian
authors.forename: A G
authors.initials: AG
authors.lastname: So
authors.forename: K M
authors.initials: KM
authors.lastname: Downey
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D003854:Deoxyribonucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006678:HIV / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H; D012265:Ribonucleotides / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D012265:Ribonucleotides; D013942:Thymine Nucleotides; C057287:3'-azido-3'-deoxythymidine 5'phosphate; D015215:Zidovudine; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H