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NLM AIDSLINE

Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain.




 

J Virol. 1993 Jul;67(7):4037-49. Unique Identifier : AIDSLINE

We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.

Amino Acid Sequence Base Sequence Cloning, Molecular HIV-1/*ENZYMOLOGY Molecular Sequence Data Oligodeoxyribonucleotides/CHEMISTRY Ribonuclease H, Calf Thymus/CHEMISTRY/*ISOLATION & PURIF RNA-Directed DNA Polymerase/*CHEMISTRY RNA, Transfer, Lys/METABOLISM RNA, Viral/METABOLISM Structure-Activity Relationship Support, Non-U.S. Gov't Support, U.S. Gov't, P.H.S. JOURNAL ARTICLE



 




Information in this article was accurate in September 30, 1993. The state of the art may have changed since the publication date. This material is designed to support, not replace, the relationship that exists between you and your doctor. Always discuss treatment options with a doctor who specializes in treating HIV.