We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16 It is well recognized that regulation of enzyme activity may be achieved by several distinct mechanisms. In recent years evidence has accumulated implicating an enzymatic interconversion of active and inactive forms of enzymes as a major regulatory control mechanism. The activity of an increasing number of enzymes may be regulated and modulated through phosphorylation or dephosphorylation involving the coordinated action of protein phosphokinases and phosphoprotein phosphatases on their enzyme substrates (1-7). The opportunities that such systems provide for coordinated control of enzymic activity are amply illustrated by the now well-established phosphorylative and functional modifications of glycogen synthetase and glycogen phosphorylase (2, 3).There is evidence that infection and transformation of susceptible cells by oncogenic RNA viruses, such as Rous sarcoma virus (RSV), is critically dependent upon the activity of the reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) (8,9). In view of this finding, it is of importance to determine whether or not the enzymatic activity of the reverse transcriptase may be altered and modified and whether the modification occurs as a consequence of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) action. In the present communication we wish to report that protein phosphokinase isolated from RSV-transformed chick embryo fibroblasts can enhance reverse transcriptase activity of Rous sarcoma virus and that the increase in activity can be reversed through the action of phosphatase. Cells and Viruses. Schmidt-Ruppin RSV, subgroup D, (SR-RSV-D) was grown in type c/o chick embryo fibroblasts and purified as described (11). Two weeks after infection by SR-RSV-D, the transformed cells were removed with scraping, washed with a solution of 0.14 M NaCl/0.1 M sodium phosphate, pH 7.2, and pelleted by centrifugation at 1000 X g. The recovered cells were used for the isolation of protein kinase.Enzyme Preparations. Protein kinase from the cytosol (105,000 X g supernatant fraction) of RSV-transformed cells was isolated and purified as described (6). After DEAE-cellulose chromatography, the pooled fractions containing protein kinase activity were additionally purified by Sephadex G-200 gel filtration (elution buffer: 0.05 M Tris buffer, pH 7.2, containing 2 mM MgCl2, and 1 mM 2-mercaptoethanol) followed by preparative isoelectric focusing (12). The protein kinase preparation used in these studies had a specific activity of 11.6 pmol of 32P incorporated/mg of protein per min using protamine sulfate as substrate. Addition of cyclic AMP (1 1,M) to this kinase preparation resulted in a 1.54-fold stimulation of the kinase activity.Reverse transcriptase from RS-RSV-D was purified by the method of Grandgenett et al. (13). The major peak of r...
Recombinant human adenoviruses (Ads) that replicate in the intestinal tract offer a novel, yet practical, means of immunoprophylaxis against a wide variety of viral and bacterial pathogens. For some infectious agents such as human immunodeficiency virus (HIV), the potential for residual infectious material in vaccine preparations must be eliminated. Therefore, recombinant human Ads that express noninfectious HIV or other microbial proteins are attractive vaccine candidates. To test such an approach for HIV, we chose an experimental model of AIDS based on simian immunodeficiency virus (SIV) infection of macaques. Our data demonstrate that the SIV Env gene products are expressed in cultured cells after infection with a recombinant Ad containing both SIV env and rev genes. An E3 deletion vector derived from a mutant of human Ad serotype 5 that efficientiy replicates in both human and monkey cells was used to bypass the usual host range restriction
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