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- Title
Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue.
- Authors
HERMAN, Brian D.; SLUIS-CREMER, Nicolas
- Abstract
EFV (efavirenz) and ß-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 ß-thujaplicinol binds in the RNase H active site and inhibits it. We have used pre-steadystate kinetic analyses to gain an insight into the mechanism by which EFV and a ß-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 polymeraseindependent 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 polymeraseindependent cleavages. Consistent with this model, we show antagonism between EFV and 19616.
- Subjects
RIBONUCLEASE kinetics; SCISSION (Chemistry); HIV-1 glycoprotein 120; CYCLOHEPTATRIENES; REVERSE transcriptase; EFAVIRENZ; DNA primers
- Publication
Biochemical Journal, 2013, Vol 455, Issue 2, p179
- ISSN
0264-6021
- Publication type
Article
- DOI
10.1042/BJ20130850