ABSTRACT
The physiological significance of casein kinase II (CK-II) on the protease (PR) activity of recombinant HIV-1 PR (rPR) was biochemically investigated in vitro. We found that (i) the purified rPR (p11) functions as a phosphate acceptor of CK-II; (ii) the PR activity of rPR is stimulated approximately 2.9-fold after its full phosphorylation by recombinant human CK-II (rhCK-II) in a manner similar to that observed for recombinant HIV-1 reverse transcriptase (rRT); and (iii) this stimulation is completely inhibited by two polyphenol-containing anti-oxidant compounds [quercetin and epigallo-catechin gallate (EGCG)] at 0.1 microM or a glycyrrhetinic acid derivative (oGA) and catechin at 10 microM without significant effect on the PR activity of rPR. These results suggest that (i) CK-II may be a host mediator responsible for stimulation of PR and RT in HIV-1-infected cells; and (ii) the selective inhibition of the CK-II-mediated stimulation of HIV-1 PR and RT by potent CK-II inhibitors may be involved in their anti-HIV-1 effects at the cellular level.
Subject(s)
HIV Protease/metabolism , HIV-1/enzymology , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Casein Kinase II , Chromatography, Affinity , Chromatography, Ion Exchange , Enzyme Activation , HIV Protease/isolation & purification , Kinetics , Phosphorylation , Recombinant Proteins/metabolismABSTRACT
The physiological significance of the casein kinase II (CK-II)-mediated phosphorylation of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on its three enzymatic activities [RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP) and ribonuclease H (RNase H)] was investigated in vitro. It was found that (i) the purified recombinant RT (rRT) functioned as an effective phosphate acceptor for CK-II; (ii) the RDDP, DDDP and RNase H activity of rRT was stimulated about 2.8-, 4.1- and 3.9-fold, respectively, after full phosphorylation by CK-II; and (iii) this stimulation was selectively inhibited by potent CK-II inhibitors, such as neocarzinostatin-chromophore (NCS-chrom) and three polyphenol-containing anti-oxidant compounds [quercetin, epigallocatechin gallate (EGCG) and 8-chloro-3',4',5,7-tetrahydroxyisoflavone (8C-3',4',5,7-THI)]. These results suggest that (i) CK-II may be responsible for activation of RT in HIV-1-infected cells; and (ii) the selective inhibition of CK-II-mediated activation of HIV-1 RT by potent CK-II inhibitors may be involved in the mechanism of their anti-HIV-1 effects at the cellular level.
Subject(s)
Enzyme Inhibitors/pharmacology , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , Protein Serine-Threonine Kinases/metabolism , Anti-HIV Agents/pharmacology , Antioxidants/pharmacology , Casein Kinase II , Enediynes , Enzyme Activation , Escherichia coli , Glycyrrhetinic Acid/pharmacology , HIV-1/drug effects , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Transfection , Zinostatin/analogs & derivatives , Zinostatin/pharmacologyABSTRACT
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.
