Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity

J Gen Virol. 1991 Jan;72 ( Pt 1):59-66. doi: 10.1099/0022-1317-72-1-59.

Abstract

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line
  • Chromosome Deletion
  • Cloning, Molecular
  • Endoribonucleases / genetics*
  • Endoribonucleases / isolation & purification
  • Endoribonucleases / metabolism
  • Escherichia coli / genetics
  • HIV-1 / enzymology
  • HIV-1 / genetics
  • HIV-1 / pathogenicity*
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • RNA-Directed DNA Polymerase / genetics*
  • RNA-Directed DNA Polymerase / isolation & purification
  • RNA-Directed DNA Polymerase / metabolism
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Ribonuclease H
  • Transfection

Substances

  • Recombinant Proteins
  • RNA-Directed DNA Polymerase
  • Endoribonucleases
  • Ribonuclease H