Oxygen-dependent Regulation of in VivoReplication of Simian Virus 40 DNA Is Modulated by Glucose

Abstract

Simian virus 40 (SV40)-infected CV1 cells exposed to hypoxia show an inhibition of viral replication. Reoxygenation after several hours of hypoxia results in new initiations followed by a nearly synchronous round of SV40 replication. In this communication, we examined the effect of glucose on inhibition of viral DNA replication under hypoxia. We found that glucose stimulated SV40 DNA replication under hypoxia in two different ways. First, the rate of DNA synthesis, i.e. the fork propagation rate, increased. This effect seemed to be mediated by inhibition of mitochondrial respiration by glucose (Crabtree effect). Inhibition of mitochondrial respiration probably resulted in a higher intracellular oxygen concentration and an activation of oxygen-dependent ribonucleotide reductase, which provides the precursors for DNA synthesis. This glucose effect was consequently strongly dependent on the strength of hypoxia and the extent of intracellular respiration; hypoxic gassing with 10 ppm instead of 200 -400 ppm O 2 or treatment of hypoxic cells with a mitochondrial uncoupler (carbonyl cyanide m-chlorophenylhydrazone) reduced the glucose effect on replication, whereas antimycin A, an inhibitor of respiration, increased it. The second effect of glucose concerned initiation, i.e. stimulation of unwinding of the viral origin. This effect was not influenced by the strength of hypoxia or the extent of cellular respiration and seemed, therefore, not to be mediated through a Crabtree effect. No evidence for a direct correlation between the cellular ATP concentration and the extent of SV40 replication under hypoxia was found. The effect of glucose on replication under hypoxia was not restricted to SV40-infected CV1 cells but was also detectable in HeLa cells. This suggests it to be a mechanism of more general validity.DNA replication in mammalian cells is subject to a fast acting regulation that depends on the O 2 tension in the cellular environment. This regulation results in inhibition of cellular replication when the concentration of O 2 falls below 0.1%. Regulation of cellular replication by O 2 has first been demonstrated for Ehrlich ascites cells (1-4). Further studies revealed that it is also valid for HeLa and CCRF cells (5), suggesting it to be a mechanism of general importance that adapts the cellular DNA replication to the supply of O 2 and other nutrients. This seems to be of particular significance during embryonic growth, wound healing, or tumor cell propagation. Inhibition of replication under hypoxia primarily affects replicon initiation. Additionally, the rate of DNA chain growth is frequently reduced. Readmission of O 2 after several hours of hypoxia reverses the suppression of DNA replication within a few minutes. This remarkably fast response suggests that the O 2 -dependent replication control acts very directly on the replication apparatus.The molecular mechanism of the oxygen-dependent replication control is largely obscure. A reduction of the intracellular concentrations of deoxynucleoside triphosphates, e...

Replication and Cytopathic Effect of Oncolytic Vesicular Stomatitis Virus in Hypoxic Tumor Cells In Vitro and In Vivo

“…We reasoned that VSV replication may be able to adapt to cells under hypoxic stress. In addition, because VSV is an RNA virus that produces its own RNA polymerase, it should not be sensitive to the hypoxia-induced inhibition of DNA and RNA synthesis in the way that DNA viruses are (33,34). Our results indicate that there was an early effect of hypoxia that inhibited the translation of viral proteins, but the virus appeared to adapt to the hypoxic environment and overcome this inhibition as the infection progressed.…”

“…One is whether this property is unique to VSV or whether other oncolytic viruses such as adenovirus, Newcastle disease virus, or reovirus share the same ability to replicate under low-oxygen conditions. Based on work with simian virus 40 showing that hypoxia blocks viral DNA replication (33,34), we believe that DNA viruses will face additional challenges to successful replication in hypoxia because of the inhibition of DNA synthesis. In contrast, RNA viruses like VSV, Newcastle disease virus, and reovirus may, as a class, be capable of surmounting the cellular adaptation to hypoxia to infect and kill cells.…”

Genetic Evidence for a Link Between Glycolysis and DNA Replication

“…Third, DNA synthesis takes place in the reductive phase of a metabolic respiration/reduction cycle in Saccharomyces cerevisiae [18], [19]. Fourth, DNA synthesis is stimulated by glucose in SV40 and in HeLa cells grown in hypoxia [20]. Fifth, mutations in glycolytic genes encoding the enolase (termed thereafter Eno), the phosphoglycerate kinase (Pgk) or the glucokinase suppress a thermosensitive (Ts) mutation in the S. cerevisiae MCM1 protein [21].…”

Re‐oxygenation of hypoxic simian virus 40 (SV40)‐infected CV1 cells causes distinct changes of SV40 minichromosome‐associated replication proteins

“…In a recent study, we have shown that glucose in millimolar concentrations prevents hypoxia‐induced inhibition of SV40 DNA replication in infected CV1 cells [62]. As we have outlined in this study, though O 2 and glucose are main substrates for cellular ATP generation, it seems unlikely that ATP shortage is responsible for inhibition of SV40 replication under hypoxia.…”