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Aspects of thymidine metabolism and function in cultured mammalian cells infected with herpes simplex virus type 1.

Summary
We have investigated aspects of thymidine metabolism and function in cultured mammalian cells infected with herpes simplex virus under conditions in which virus DNA synthesis was either permitted, or was prevented by inhibiting the virus-induced DNA polymerase with phosphonoacetate. In 30 min pulse-labelling experiments the rates of [3H]thymidine transport into cells and its subsequent phosphorylation both reached maximum values about 6 h after infection. During the next 17 h these rates remained relatively constant in the absence of phosphonoacetate but declined to about 50% of the 6 h value in the presence of this inhibitor. When the radioactive thymidine was present continuously throughout the infection the accumulated intracellular acid-soluble radioactivity reached maximum values at about 9 h. In the absence of phosphonoacetate this declined thereafter, but it remained relatively constant when virus DNA synthesis was prevented. This suggests some established equilibrium between the continuing entry of thymidine into cells and its subsequent removal by excretion. Excretion of thymidine-derived radioactivity was initially established by determining the fate of isotopically labelled host cell DNA. During 24 h of infection about 50% of the host cell DNA was rendered acid-soluble and of this 60 to 70% was excreted from the cells. High pressure liquid chromatographic analysis of the intracellular acid-soluble radioactivity showed only phosphorylated thymidine derivatives, whereas the excreted radioactivity was exclusively in thymidine. Excretion of intracellular radioactive molecules derived directly from [3H]thymidine in the medium was also observed, but only when virus DNA synthesis was inhibited with phosphonoacetate.
Simple kinetic studies with the purified virus-induced DNA polymerase showed that a straight line double-reciprocal plot was obtained when dGTP was used as the limiting substrate (K
m = 0.8 µm, V
max = 330 nmol dGTP incorporated/10 min) but that a biphasic plot was obtained when dTTP was limiting (K
m1 = 0.5 µm, V
max1 = 208 nmol dTTP incorporated/10 min; K
m2 = 5 µm, V
max2 = 370 nmol dTTP incorporated/10 min).