Biochemical studies on adenovirus multiplication: III. Requirement for DNA synthesis

Abstract

The metabolic inhibitor 5-fluorodeoxyuridine (FUDR) was used in a study of the role of DNA synthesis in the multiplication of type 2 adenovirus in suspension cultures of KB cells. Treatment with 10 −6 M FUDR stopped DNA synthesis in infected and uninfected cells and prevented virus multiplication in infected cells, while permitting the synthesis of protein, RNA, and acid-soluble nucleotides in infected and uninfected cells. It is concluded that DNA synthesis is essential for adenovirus multiplication. The time course of viral DNA synthesis was examined by two procedures: (1) FUDR was added at various times after infection and the final virus yield determined. The final virus yield is a measure of the viral DNA synthesized prior to the time of addition of FUDR. When FUDR was added up to 7 hours after infection, no virus was detected; when it was added after this time virus was formed in increasing amounts. These results indicate that viral DNA is synthesized from about 7 to 21 hours after infection; infectious virus is made from about 14 to 28 hours. (2) The FUDR-inhibition of adenovirus multiplication was reversed at various times after infection and virus growth curves were determined. Thymidine at 10 −5 M completely reversed the inhibition of virus multiplication by 10 −6 M FUDR. When thymidine was added within the first 7 hours after infection to cultures treated with FUDR at the time of infection, the growth curves obtained were identical to those of noninhibited cultures; when thymidine was added at 8–15 hours after infection, virus maturation in each case began about 7 hours later and proceeded at an accelerated rate. One may conclude the following from these experiments: (a) DNA synthesis is not required for viral development during the first 7 hours of the eclipse period; (b) the synthesis of viral DNA begins at 7 hours after infection; (c) about 7 hours of DNA synthesis is required before virus maturation is initiated.