An early temperature-sensitive event which prevents the replication of variola-virus DNA in HeLa cells at 40° was studied in ‘temperature-shift’ experiments using 5-fluorodeoxyuridine or 5-bromodeoxyuridine. ‘Shift-up’ from 35° to 40° suppressed the replication of virus DNA, rapidly halting it even after it had begun at 35°. ‘Shift-down’ from 40° to 35° permitted the replication of virus DNA after a delay of not more than 2 hr. These results were confirmed in studies of the incorporation of [H]thymidine into DNA in the cytoplasmic fraction. It was also shown that the incorporation which followed ‘shift-down’ from 40° could be inhibited by adding -fluorophenylalanine at the time of shift. When cytoplasmic fractions from cells incubated at different temperatures were examined for enzyme activities, there was a marked increase in thymidine kinase activity in infected cells both at 35° and at 40°. DNA polymerase activity was increased five- to sixfold in infected cells at 35° but was not increased at 40° or in the presence of -fluorophenylalanine. The increased polymerase activity in infected cells at 35° was unstable at 40° both and , in contrast to its greater stability at 35°. The behaviour of this enzyme was thought to explain the observed temperature-sensitivity of the replication of variola virus DNA.


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