Infection of monkey kidney (BSC-40) cells with vaccinia virus strain WR resulted in a marked increase in
ribonucleoside diphosphate reductase (EC 1.17.4.1) activity as measured by
CDP reduction in cell-free extracts. After a synchronous
infection, increased activity was detected at 2 h, peaked at 4 to 5 h, and then declined between 6 and 8 h to the endogenous cellular level. The induction, detectable at 0.5 PFU/cell, correlated strongly with multiplicity of
infection to 10 PFU/cell and continued to increase to 50 PFU/cell. It paralleled the previously described induction of
viral DNA polymerase and
thymidine kinase, suggesting that the
reductase may also be a product of early transcription of the viral genome. The inhibition of
DNA synthesis throughout
infection resulted in prolonged accumulation of
reductase activity and delayed and incomplete down-regulation at 8 h, suggesting that repression involves late functions. Rescue of
fluorodeoxyuridine-inhibited
DNA synthesis with exogenous
thymidine restored the normal pattern. Preferential association of the induced
reductase with the cytoplasmic sites of vaccinia virus DNA replication (
virosomes) was not detected. The induced
enzyme is similar in several respects to other eucaryotic
ribonucleotide reductases, but is distinct from host cell
reductase in response to certain modulators of
reductase activity (M. B. Slabaugh and Christopher K. Mathews, J. Virol. 52:501-506, 1984). Full activity required an activator, exogenous reducing equivalents, and
iron.
Hydroxyurea,
EDTA, dATP, and
dTTP inhibited
CDP reduction, setting this
reductase apart from T4
reductase, which is not inhibited by dATP, and from herpesvirus
reductase, which requires no activation and is insensitive to deoxyribonucleoside
triphosphate inhibition.