Abstract
SV40 (simian virus 40)-infected CV1 cells were permeabilized with Staphylococcus aureus alpha-toxin for small molecules (<2 kDa) in a medium that supports DNA replication. Incorporation of [alpha-32P]dATP was shown to proceed at an essentially constant rate for at least 1 h. 32P-labelled DNA replication intermediates and products were analysed by alkaline sucrose density centrifugation. The results suggested that SV40 DNA replication in alpha-toxin-permeabilized CV1 cells occurred essentially as in vivo. After bromodeoxyuridine 5'-triphosphate-labelling and isopycnic banding, significant amounts of DNA density-labelled in both strands were detected from 110 min of permeabilization onwards, indicating repeated rounds of viral DNA replication in the permeabilized cells. Incubation of permeabilized SV40-infected cells under hypoxic culture conditions caused inhibition of SV40 DNA replication. As seen in unpermeabilized cells, SV40 DNA replication was inhibited at the stage of initiation. The inhibition of DNA replication induced by hypoxia was mimicked by AA (antimycin A), an inhibitor of mitochondrial respiration, and also by the replacement of glutamate, a substrate of mitochondrial respiration, by Hepes in the permeabilization medium. Inhibition of DNA replication was not mediated by intracellular ATP depletion. AA also inhibited SV40 DNA replication in unpermeabilized, normoxically incubated cells. Moreover, as in hypoxically incubated cells, the addition of glucose to SV40-infected cells incubated for several hours with AA induced a burst of new initiations followed by a nearly synchronous round of viral DNA replication. Taken together, these results indicate that mitochondria are involved in the oxygen-dependent regulation of SV40 DNA replication.