Mutational analysis of the DNA polymerase and ribonuclease H activities of human immunodeficiency virus type 2 reverse transcriptase expressed in Escherichia coli

Virology. 1991 Jan;180(1):339-46. doi: 10.1016/0042-6822(91)90038-d.

Abstract

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a polypeptide with an apparent molecular weight of 68 kDa. The HIV-2 reverse transcriptase (RT) made in E. coli is soluble in bacterial extracts and possesses both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities typical of retroviral RTs. The HIV-2 RT expression clone was used to generate mutations in HIV-2 RT. There is a strong correlation between the effects of individual mutations on the DNA polymerase and RNase H activities. Mutations that profoundly affect the two catalytic functions are not clustered in any particular region of the polypeptide. Those few mutations that selectively affect either the RNase H or the DNA polymerase suggest that, like other retroviral RTs, the DNA polymerase is associated with the amino-terminal portion of HIV-2 RT and the RNase H with the carboxy-terminal portion. Genetically, the HIV-2 RT resembles the HIV-1 RT more closely than it resembles Moloney murine leukemia virus RT. The two catalytic functions of Moloney murine leukemia virus RT can be separately expressed in active form by molecular cloning; those of HIV-1 and HIV-2 RT cannot.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • DNA Mutational Analysis
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • Endoribonucleases / genetics*
  • Endoribonucleases / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Gene Expression
  • HIV-1 / enzymology
  • HIV-2 / enzymology*
  • HIV-2 / genetics
  • Molecular Sequence Data
  • Mutagenesis
  • Peptide Biosynthesis
  • Plasmids / genetics
  • RNA-Directed DNA Polymerase / genetics*
  • RNA-Directed DNA Polymerase / metabolism
  • Ribonuclease H
  • Sequence Homology, Nucleic Acid

Substances

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