Enzyme induction in SV40-infected green monkey kidney cultures

Abstract

After infection of primary cultures of green monkey kidney cells or of an established green monkey kidney cell line (CV-1) with SV40, thymidine kinase activity was induced. Increased enzyme activity was first detectable 12–16 hours after inoculation and was 4–15 times greater at 28–48 hours in infected than in noninfected cultures. Thymidine kinase has been partially purified from noninfected and SV40-infected cells. With deoxyuridine (UdR) as substrate, the Michaelis constant of the enzyme from infected cells was 2–3 times greater than that from noninfected cells. However, the kinetics of inactivation of the enzymes were about the same at 38 °. 1-β- d-Arabinofuranosylcytosine (ara-C) inhibited the incorporation of tritiated thymidine (H 3-TdR) into the deoxyribonucleic acid (DNA) of noninfected or SV40-infected monkey kidney cultures by 95% or more. Ara-C treatment increased greatly the thymidine kinase activity of noninfected monkey kidney cultures. The Michaelis constant of thymidine kinase purified from ara-C treated CV-1 cultures was identical to that from untreated cultures. Ara-C did not prevent the induction of thymidine kinase by SV40. Indeed, the effects of ara-C and SV40 on enzyme induction were approximately additive in drug treated and infected cultures. Bromodeoxyuridine (BUdR) treatment did not prevent thymidine kinase induction by SV40, although virus growth was inhibited by 99.9%. Unlike ara-C, BUdR did not have a stimulating effect on thymidine kinase activity in noninfected cells. Cycloheximide inhibited the incorporation of radioactive amino acids into proteins of monkey kidney cells. Cycloheximide prevented thymidine kinase induction when added to cultures early after SV40 infection and arrested further enzyme induction when added after thymidine kinase induction had begun. A gradual twofold increase in thymidylate synthetase activity occurred in SV40-infected cultures but thymidine 5′-monophosphate (TMP) kinase and uridine kinase activities increased by less than 50%. Radioautographic and biochemical experiments on the incorporation of radioactive precursors into DNA demonstrated that the percentage of cells with labeled nuclei and the uptake of either tritiated deoxyadenosine or H 3-TdR into DNA was sharply increased beginning at about 16–20 hours after infection. The total DNA content of SV40-infected cultures was also increased.