title: Expression of an Mg2+-dependent HIV-1 RNase H construct for drug screening.
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.
authors.affiliation: Programa de Virología, Instituto de Ciencias Biomédicas, Facultad de Medicina Universidad de Chile, Independencia 1027, Santiago, Chile.
authors.forename: Richard V
authors.initials: RV
authors.lastname: Farias
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Vargas
authors.forename: Andres E
authors.initials: AE
authors.lastname: Castillo
authors.forename: Beatriz
authors.initials: B
authors.lastname: Valenzuela
authors.forename: Marie L
authors.initials: ML
authors.lastname: Coté
authors.forename: Monica J
authors.initials: MJ
authors.lastname: Roth
authors.forename: Oscar
authors.initials: O
authors.lastname: Leon
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology*; D001483:Base Sequence; D004353:Drug Evaluation, Preclinical; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D012343:RNA, Transfer / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017423:Sequence Analysis, RNA
chemical_list: D019380:Anti-HIV Agents; D018894:Reverse Transcriptase Inhibitors; D012343:RNA, Transfer; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H; D008274:Magnesium