George N. Jorgensen

Thymidine Kinases Induced by Avian and Human Herpesviruses

Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation experiments were carried out to characterize thymidine (dT) kinase isozymes induced by herpesvirus of turkeys and infectious laryngotracheitis virus in the cytosol fraction of infected chick cells. The avian herpesvirus dT kinases differed from chick cytosol dT kinase in electrophoretic mobility, isoelectric point and phosphate donor specificity. The avian herpesvirus dT kinases resembled chick mitochondrial dT kinase in electrophoretic mobilityy and isoelectric point, but exhibited larger sedimentation coefficients. The avian herpesvirus enzymes closely resembled dT kinases induced by human herpes simplex viruses types 1 and 2.

Purification of vaccinia virus-induced thymidine kinase activity from [35S]methionine-labeled cells

Abstract Vaccinia virus thymidine kinase (TK) activity was purified from the cytosol of [ 35 S]methionine-labeled, virus-infected LM(TK-) mouse fibroblast cells. The purification procedure entailed (i) selective binding of TK activity to a Sepharose 4B-5′-amino-5′-deoxythymidine gel and elution of the enzyme from the enzyme-gel complex, (ii) preparative polyacrylamide gel electrophoresis (PAGE), and (iii) glycerol gradient centrifugation of the purified vaccinia virus TK activity. [ 35 S]Methionine-labeled cytosol fractions from uninfected cells were also mixed with nonlabeled vaccinia virus TK and purified by the same procedure to determine whether cellular proteins copurified with vaccinia virus TK. The purified vaccinia virus TK activity was adsorbed to an immunoadsorbent, made by coupling immunoglobulins (IgG) from antivaccinia virus TK rabbit antisera with CNBr-activated Sepharose 413. After elution of the labeled polypeptides from the immunoadsorbent, the eluates, as well as samples from earlier purification steps, were analyzed by SDS-polyacrylamide slab gel electrophoresis and autoradiography. Radioactive vaccinia virus TK that had been partially purified by analytical disc PAGE and by isoelectric focusing in polyacrylamide gels was also analyzed by SDS-polyacrylamide slab gel electrophoreses and autoradiography. The results of these experiments suggest that vaccinia virus TK consists of two subunits with molecular weights of about 40,000–42,000. Induction of the 40,000- to 42,000-dalton polypeptide was also observed in cells infected by vaccinia virus mutant 1004B, suggesting that this mutant induces the formation of an enzymatically inactive TK polypeptide.

Subcellular Localization and Properties of Thymidine Kinase from Adenovirus-infected Cells

Summary The principal cytosol thymidine kinase activity of African green monkey kidney cells increased approximately threefold at 21 to 29 h after infection with adenovirus type 5. Disc polyacrylamide gel electrophoresis (disc PAGE) analyses showed that the electrophoretic mobility relative to the tracking dye (Rm) of the cytosol thymidine kinase from both mock-infected and adenovirus type 5-infected cells was about 0.23. The cytosol thymidine kinase from normal cells also resembled the cytosol enzyme from infected cells with respect to phosphate donor specificity and sedimentation coefficient. Mitochondria from normal and virus-infected cells contained a cytosol-like enzyme and, in addition, a distinctive mitochondrial isozyme exhibiting an Rm of about 0.6 and a smaller sedimentation coefficient than the cytosol enzyme. The activity of the mitochondrial-specific isozyme of thymidine kinase (Rm = 0.6) was not significantly increased by virus infection. The ratio of the two thymidine kinase activities found in mitochondria also was not markedly changed by virus infection. The results suggest that adenovirus type 5 infection reactivates an inactive molecular form of cytosol thymidine kinase or derepresses the synthesis of the cytosol enzyme, but not that of the mitochondrial-specific thymidine kinase. Adenovirus infection does not alter the electrophoretic mobilities of the cytosol and mitochondrial thymidine kinases.

Biochemical and serological properties of the thymidine-phosphorylating enzymes induced by herpes simplex virus mutants temperature-dependent for enzyme formation

Abstract Evidence is presented in support of the hypothesis that: (i) an enzyme induced by wild-type herpes simplex virus type 1 (HSV-1) is a deoxypyrimidine kinase with a single active site for the phosphorylation of both thymidine (TdR) and deoxycytidine (CdR); (ii) enzymes induced by HSV-1 mutants that are temperature dependent for enzyme induction are altered with respect to nucleoside acceptor specificity and antigenicity; and (iii) altered polypeptides are synthesized in mutant virus-infected cells at both the permissive (31°) and restrictive (37.5°) temperatures. HSV-1 mutants B2010 and B2015 induce less TdR-phosphorylating activities at 31° than wild-type HSV-1 and very low but detectable enzyme activities at 37.5°. The TdR-phosphorylating activities induced by the mutant viruses at 31° have the same electrophoretic mobilities as that of the wild-type HSV-1-induced enzyme but, unlike the wild-type enzyme, the mutant enzymes lack CdR-phosphorylating activity. Immunoglobulin G (IgG) prepared from rabbits immunized with cytosol extracts from rabbit kidney cells infected with wild-type HSV-1 inhibit the TdR-phosphorylating activities induced by wild-type and mutant viruses, as well as the CdR-phosphorylating activity induced by wild-type HSV-1. Extracts purified from cells infected at 31° with wild-type and mutant HSV-1 blocked the neutralizing activity of anti-HSV-1 IgG for the CdR- as well as the TdR-phosphorylating activities of wild-type HSV-1 enzyme, despite the lack of CdR-phosphorylating activity of the mutant enzyme. However, the mutant virus-infected cell extracts were less effective than wild-type extracts in serum blocking activity. Extracts from cells infected with mutant B2015 at 37.5° did not exhibit serum blocking power.

Gel Electrophoresis, Isoelectric Focusing, and Localization of Thymidine Kinase in Normal and Simian Virus 40-Infected Monkey Cells

Following SV40 infection of CV-1 monkey cells, cytosol thymidine (dT) kinase activity increased 4- to 10-fold. This enzyme was not significantly different from the cytosol dT kinase of uninfected cells with respect to mobility, isoelectric point, sedimentation coefficient, and phosphate donor specificity. Monkey mitochondria contain a distinctive dT kinase isozyme localized in the mitochondrial matrix. The distinctive mitochondrial isozyme had a higher electrophoretic mobility, lower isoelectric point, and smaller sedimentation coefficient than the cytosol dT kinase, and could utilize either ATP or UTP as phosphate donor; it did not increase after SV40 infection. Mitochondria also contained a cytosol-like dT kinase which was enhanced in infected cells. These findings suggest that SV40 infection derepresses the normal cytosol dT kinase of host cells.