Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue.
EFV (efavirenz) and β-thujaplicinol [2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one] have contrasting effects on the RNase H activity of HIV-1 RT (reverse transcriptase). EFV binds in the non-nucleoside inhibitor-binding pocket and accelerates this activity, whereas β-thujaplicinol binds in the RNase H active site and inhibits it. We have used pre-steady-state kinetic analyses to gain an insight into the mechanism by which EFV and a β-thujaplicinol analogue [19616 (2,7-dihydroxy-2,4,6-cyclo-heptatrien-1-one)] modulate RT RNase H activity. Our data show that EFV and 19616 have no effect on polymerase-dependent RNase H cleavages. However, both compounds significantly affected the rates of polymerase-independent RNase H cleavages. In regard to the latter, we found no evidence that the bound RNA/DNA template/primer substrate restricted 19616 from interacting with RT. In light of these data, we propose a model in which 19616 binds to the RNase H active site of RT after the primary polymerase-dependent RNase H cleavage has occurred and stabilizes the 3'-end of the DNA primer in the polymerase active site thus blocking the enzyme's ability to carry out the polymerase-independent cleavages. By contrast, EFV destabilizes the 3'-end of the DNA primer in the DNA polymerase active site and promotes RT-mediated polymerase-independent cleavages. Consistent with this model, we show antagonism between EFV and 19616.
*Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
Brian D
BD
Herman
Nicolas
N
Sluis-Cremer
D000480:Alkynes; D019380:Anti-HIV Agents / Q000494:pharmacology*; D048588:Benzoxazines / Q000494:pharmacology*; D001665:Binding Sites; D003521:Cyclopropanes; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014334:Tropolone / Q000031:analogs & derivatives* / Q000494:pharmacology
D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D018894:Reverse Transcriptase Inhibitors; C497842:beta-thujaplicinol; D014334:Tropolone; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; C098320:efavirenz