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Antimicrob Agents Chemother. 2011 Oct;55(10):4735-41. doi: 10.1128/AAC.00658-11. Epub 2011 Jul 18.

Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening.

Author information

1
Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina Universidad de Chile, Independencia 1027, Santiago, Chile.

Abstract

A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg(2+)-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg(2+)-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar K(m)s for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

PMID:
21768506
PMCID:
PMC3186996
DOI:
10.1128/AAC.00658-11
[Indexed for MEDLINE]
Free PMC Article

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