Biochemical characterization of recombinant HIV-1 reverse transcriptase (rRT) as a glycyrrhizin-binding protein and the CK-II-mediated stimulation of rRT activity potently inhibited by glycyrrhetinic acid derivative.
By means of successive Mono Q and glycyrrhizin (GL)-affinity column chromatography (HPLC), recombinant HIV-1 RT (rRT) was purified to apparent homogeneity from the Superdex 200 pg fraction of the crude protein extract of E. coli BL21 transfected with pET 21a(+)/HIV-1 PR-RT. It was found that (i) rRT functioned as an effective phosphate acceptor for recombinant human casein kinase II (rhCK-II) in vitro; (ii) this phosphorylation was inhibited by anti-HIV-1 substances [a glycyrrhetinic acid derivative (oGA) and quercetin] and a high dose (100 microM) of GL; (iii) RNA-dependent DNA polymerase (RDDP) activity was stimulated about 2.5-fold after full phosphorylation of rRT by rhCK-II; and (iv) oGA as well as NCS-chromophore effectively prevented the CK-II-mediated stimulation of RDDP activity. These results suggest that the anti-HIV-1 effect of oGA may be involved in the selective inhibition of the CK-II-mediated stimulation of HIV-1 RT at the cellular level.
Harada
S
S
Laboratory of Genetical Biochemistry, Kitasato University School of Allied Health Sciences, Sagamihara, Japan.
Maekawa
T
T
Haneda
E
E
Morikawa
Y
Y
Nagata
N
N
Ohtsuki
K
K
D019380:Anti-HIV Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D047390:Casein Kinase II; D002851:Chromatography, High Pressure Liquid; D053281:Enediynes; D004789:Enzyme Activation; D004926:Escherichia coli; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D009353:Zinostatin / Q000031:analogs & derivatives / Q000494:pharmacology
D019380:Anti-HIV Agents; D002352:Carrier Proteins; D053281:Enediynes; D011994:Recombinant Proteins; C086010:glycyrrhizin-binding proteins; C032372:neocarzinostatin chromophore; D009353:Zinostatin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D006034:Glycyrrhetinic Acid