Construction of the chimeric reverse transcriptase of simian immunodeficiency virus sensitive to nonnucleoside reverse transcriptase inhibitor.
A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors. The nonnucleoside RT inhibitors are quite specific for HIV-1 RT but not human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) RT. We have investigated the property of SIV/HIV-1 chimeric viruses in which portions of SIV(MAC) RT were exchanged with the corresponding domain of HIV-1 RT; amino acids 176-190, 176-383 and 176-495 of HIV-1 RT. The chimeric virus, which was substituted amino acids 176-190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors [nevirapine, HEPT derivative (E-EBU-dM) and TIBO derivative (R82913)]. To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the Km, and Vmax values, and the Ki value of HEPT derivative calculated for the DNA polymerase activity. This study reveals that amino acids 176-190 of SIV(MAC) RT were important for the enzymatic activity and the SIV/HIV-1 chimeric RT, which had amino acids 176-190 of HIV-1, was sensitive to the nonnucleoside RT inhibitor.
Isaka
Y
Y
Discovery Research Laboratories 1, Shionogi & Co., Ltd., Settsu, Osaka, Japan.
Sato
A
A
Kawauchi
S
S
Suyama
A
A
Miki
S
S
Hayami
M
M
Fujiwara
T
T
D000595:Amino Acid Sequence; D001569:Benzodiazepines / Q000494:pharmacology; D002478:Cells, Cultured; D054303:HIV Reverse Transcriptase / Q000187:drug effects / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology / Q000254:growth & development; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D019829:Nevirapine / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000187:drug effects* / Q000235:genetics; D016865:Reassortant Viruses / Q000201:enzymology / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000187:drug effects; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D015302:Simian Immunodeficiency Virus / Q000201:enzymology* / Q000254:growth & development; D014162:Transfection; D014498:Uracil / Q000031:analogs & derivatives* / Q000494:pharmacology
C112359:1-ethoxymethyl-5-ethyl-6-(3,5-dimethylbenzyl)uracil; D007093:Imidazoles; D011993:Recombinant Fusion Proteins; D018894:Reverse Transcriptase Inhibitors; D001569:Benzodiazepines; C067413:R-82913; D014498:Uracil; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase