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

J. S. Smith, M. J. Roth

Research output: Contribution to journalArticlepeer-review

66 Scopus citations

Abstract

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 Ni2+- 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 tRNA3/(Lys) 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.

Original languageEnglish (US)
Pages (from-to)4037-4049
Number of pages13
JournalJournal of virology
Volume67
Issue number7
DOIs
StatePublished - 1993

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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