Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization

J Biol Chem. 1994 Oct 21;269(42):26472-8.


"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • DNA-Directed DNA Polymerase / metabolism
  • HIV Reverse Transcriptase
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • HIV-1 / pathogenicity
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • RNA-Directed DNA Polymerase / chemistry
  • RNA-Directed DNA Polymerase / metabolism*
  • Ribonuclease H / physiology
  • Structure-Activity Relationship
  • Tryptophan


  • Tryptophan
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase
  • Ribonuclease H