Deoxythymidine Kinase Induced in HeLa TK-Cells by Herpes Simplex Virus Type I and Type II AND

Deoxythymidine kinase activities were induced in HeLa TK-(deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg*+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45’ and 4”. The deoxycytidine kinase activity present in the purified preparations was inactivated

From the Department of Experimental Therapeutics and Grace Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 Deoxythymidine kinase activities were induced in HeLa TK-(deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography.
Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg*+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45' and 4". The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg'+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.
Deoxythymidine kinase has been found to increase in various mammalian cells after infection with herpes simplex virus type I or type II. The induced dThd kinases differ from the host cell dThd kinases with regard to immunogenicity (l-3), molecular weight (4, 5), substrate specificity (3, 4), electrophoretic mobility (4), and thermostability (5). Host cell mitochondrial and HS-I1 virus-induced dThd kinases are able to use ATP, GTP, UTP, and CTP as phosphate donors, whereas the host cytosol dThd kinase can use only ATP as the phosphate donor (4). The host mitochondrial enzyme is relatively more sensitive to dCTP inhibition than either the HS-I virus enzyme or the host cytosol enzyme. The sedimentation coefficient of HS-II virus dThd kinase is slightly less than that of HS-I virus dThd kinase (3). The HS-II virus enzyme is *This work was supported in part by research project Grant CA-05298, core program Grant CA-13038 from the National Cancer Institute, United States Public Health Service, and research project Grant CH-29 from the American Cancer Society.
$ To whom reprint requests should be addressed. 'The abbreviations used are HS-I virus and HS-II virus, herpes simplex virus type I and type II, respectively; dThd, deoxythymidine; dCyd, deoxycytidine. somewhat less stable at 40" than the HS-I virus enzyme (5). Recently, it was suggested that the herpes-induced dThd kinase also possesses dCyd kinase activity (2, 6-8); these data were obtained with either crude or partially purified enzyme.
Because herpes type viruses can cause many human diseases and also have the potential to induce cell transformation (g-11), there is need for chemotherapeutic agents which inhibit the growth of these viruses. This laboratory has been interested in developing anti-herpes drugs by exploiting the unique properties of the herpes-induced dThd kinase. The unexpected presence of HS-I virus-induced enzyme in the nucleus, time course of induction, and the cellular distribution of the induced enzymes as well as the isolation, purification, and some properties of these enzymes are reported here.

Electrophoresis and Glycerol
Gradient Centrifugation-Electrophoresis was performed as described i n the preceding paper (16). Glycerol gradient (10 to 30%, v/v) centrifugation conditions were the same as those described by Kit and co-workers (4) except that the running time was 30 hours.
Materials-The affinity column matrix was prepared according to Kowal and Markus (17) with the modifications described i n the preceding paper (16).   Lee and Cheng (16). The samples (25 pl with protein concentration and the specific activity as indicated in Table I kinase from HS-I virus-infected cells was different from that of the purified HS-I virus dThd kinase. Relative mobility with respect to bromphenol blue for either the crude or the purified HS-II virus dThd kinase was 0.42 and for the crude or the purified HS-I virus dThd kinase, relative mobilities were 0.45 and 0.70, respectively. When the purified HS-I virus-specific dThd kinase was incubated with the 50% ammonium sulfate precipitate from mock infected cells, the electrophoretic mobility of dThd kinase was shifted back to that of the unpurified or partially purified enzyme (ammonium sulfate fractionated); the activity of the enzyme was not affected.
Glycerol Gradient Centrifugation-The results of an experiment in which SH-amino-acid-labeled purified HS-I virus or HS-II virus dThd kinase preparations were layered on top of a 10 to 30% glycerol gradient and then centrifuged for 30 hours are depicted in Fig. 5. The radioactive 3H peak coincided with the enzymatic activity peak in both cases. The recovery of the activity of purified HS-I virus or HS-II virus dThd kinase after centrifugation was 25% and 98% respectively. The sedimentation rate for both purified preparations was the same as that of the corresponding unpurified preparation, since 50 PM dThd used to stabilize dThd kinase during centrifugation is sufficient to inhibit the dCyd kinase activity in our assays,= the activity of dCyd kinase was not measured after centrifugation.
Heat Inactiuation-Purified HS-I virus and HS-II virus dThd kinase were incubated at 45" in 0.2 M Tris-HCl buffer at pH 7.5 in the presence of 2 mM Mg*+ATP, 3 mM dithiothreitol, 0.5 mg/ml of albumin, and 10% glycerol. The dThd kinase and dCyd kinase activities were measured at designated time intervals as indicated in Fig. 6, A and B. The HS-I virus dThd kinase was more unstable than the HS-II virus dThd kinase under these conditions with, a half-life of 28 min and 120 min respectively (Fig. 6A) The glycerol density gradient centrifugation conditions were the same as those described by Dr. S. Kit's laboratory (4) except that the running time was 30 hours. Hemoglobin (Hb) and pyruvate kinase (PK) were used as markers.
The dThd kinase samples (  The components of the rest of the assay were the same as described in the text. The amount of enzyme used per assay was 2 x 1O-2 unit. seems to suggest that the preparations obtained were reasonably pure. The