Altered properties of thymidine kinase after infection of mouse fibroblast cells with herpes simplex virus.

Previous studies have shown that, after infection by herpes simplex virus (HSV) or vaccinia virus, thymidine kinase activity was induced in either LM mouse fibroblast cells or LM(TK-) cells, a mutant subline lacking this enzyme activity. At 5 to 7 hr after infection, the enzyme activities induced by these viruses in the LM(TK-) cells were about three times as high as those found in exponentially growing cultures of noninfected LM cells. Mutant vaccinia virus and HSV strains have been isolated which lack enzyme-inducing activity (D. R. Dubbs and S. Kit, Virology 22:214, 1964; Virology 22:493, 1964). Partially defective HSV mutants have been obtained which fail to induce the enzyme at 37 C, but which induce about one-tenth the enzyme level attained with the wild-type virus at 31 C (D. R. Dubbs and S. Kit, Virology 25:256, 1965). The thymidine kinase partially purified from vaccinia virus-infected LM(TK-) cells differed from the enzyme from noninfected LM cells in thermal stability, serological properties, and in its Michaelis constant (Km.) for deoxyuridine (dU) (S. Kit and D. R. Dubbs, Virology 26:16, 1965). The LM celland the HSV-induced enzymes, however, were thermally inactivated at about the same rate. Data are now presented demonstrating that the kinetic properties of the HSV-induced thymidine kinase are different from those of the enzyme from either noninfected or from vaccinia virus-infected cells. Cultures ofLM and LM(TK-) cells were grown as described previously (S. Kit and D. R. Dubbs, Virology 18:274, 1962). Confluent monolayer cultures were infected with these viruses at input multiplicities of 1 to 7 plaque-forming units (PFU) per cell. The cells were harvested 6 to 7 hr after infection. Cell extracts containing thymidine kinase activity were assayed with tritiumlabeled deoxyuridine (3H-dU) as nucleoside substrate. The assay was routinely performed at three enzyme concentrations, and activity was proportional to enzyme concentration under the conditions employed. The enzyme was partially purified by the procedure previously described (S. Kit et al., Virology 29:69, 1966), except for the following modifications. (i) The centrifuged enzyme extracts (fraction S3) were first mixed for 8 min at 4 C with calcium phosphate gel (1.5 mg of gel per mg of protein in fraction S3). (ii) After centrifugation to remove the calcium phosphate gel, 0.14 g of ammonium sulfate was added slowly to the supernatant fluid. The precipitate was discarded, and an additional 0.14 g of ammonium sulfate was slowly added to precipitate the thymidine kinase activity (As 20-40 fraction). The enzyme was then dissolved in buffer solution [0.15 M KCl; 0.01 M tris(hydroxymethyl)aminomethane (Tris chloride buffer), pH 8.0; 0.003 M 2-mercaptoethanol] in one-fourth the volume of the crude-enzyme extract at a concentration of 0.24 to 0.59 mg of protein per ml of enzyme. (iii) With enzyme extracts from vaccinia virus-infected cells, the centrifuged enzyme fraction (S3) was mixed with calcium phosphate gel equivalent to 0.5 mg of gel per mg of protein. The reduction in the amount of calcium phosphate gel was necessary because the vaccinia virus-induced enzyme differed from the LM celland the HSV-induced thymidine kinases in that it was tightly adsorbed by higher amounts of calcium phosphate. By use of the modified procedure, approximately one-fifth to one-third of the total activity present in crude-enzyme extracts was recovered. The purified LM, herpes simplex-induced, and vaccinia-induced thymidine kinases, respectively, exhibited 4 to 18, 12 to 28, and 5 times greater specific activities than the crude-enzyme extracts. The specific activity of the purified vacciniainduced enzyme was 64 [expressed as millimicromoles of deoxyuridine monophosphate (dUMP) formed per milligram of protein in 10 min at 38 C]. The specific activities of the herpes simplex-