Inhibition of the RNA-directed DNA polymerase activity of a recombinant HIV-1 p51 reverse transcriptase by a p15 ribonuclease H domain.
The polymerase domain of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, called the p51 reverse transcriptase (p51 RT), was expressed in Escherichia coli. The recombinant protein also contained an N-terminal affinity tag designed to facilitate its purification by immobilized metal affinity chromatography. The purified p51 RT is a predominantly monomeric protein and it catalyses RNA-dependent DNA polymerization with poly(rA).oligo(dT) as the template.primer. Recently we have also reported the isolation of the recombinant RNAase H domain of HIV-1 RT that is enzymically active (Evans, Brawn, Deibel, Tarpley and Sharma [1991] J. Biol. Chem. 266, 20583-20585). The latter directly inhibits the RNA-dependent DNA polymerase activity of p51 RT. Kinetic experiments show that the p15 RNAase H-mediated inhibition of p51 RT is competitive with respect to the poly(rA).oligo(dT) template.primer (Ki = 320 +/- 50 nM), and it does not interfere directly with the binding of dTTP to the enzyme. Thus the kinetic behaviour is consistent with the binding of p15 RNAase H at or near the template.primer-binding site in this replicase. If the binding of the p15 RNAase H involves only a small segment of this protein, then identification of that segment may open up new opportunities towards the design of novel inhibitors of RNA-dependent DNA polymerase activity.
Evans
D B
DB
Biochemistry Research, Upjohn Laboratories, Kalamazoo, MI 49001.
Kézdy
F J
FJ
Tarpley
G
G
Sharma
S K
SK
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors; D016914:Ribonuclease H / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D011061:Poly A; C018368:poly(rA).oligo(dT); D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H