Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening.
Antimicrob Agents Chemother
; 55(10): 4735-41, 2011 Oct.
Article
in En
| MEDLINE
| ID: mdl-21768506
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
HIV-1
/
Ribonuclease H
/
Reverse Transcriptase Inhibitors
/
Anti-HIV Agents
/
HIV Reverse Transcriptase
Type of study:
Diagnostic_studies
/
Screening_studies
Language:
En
Journal:
Antimicrob Agents Chemother
Year:
2011
Document type:
Article
Affiliation country:
Chile