Hypoxia Blocks In Vivo Initiation of Simian Virus 40 Replication at a Stage Preceding Origin Unwinding

Abstract

Simian virus 40 (SV40)-infected CV1 cells transiently exposed to hypoxia show a burst of viral replication immediately after reoxygenation. DNA precursor incorporation and analysis of growing daughter strands by alkaline sedimentation demonstrated that SV40 DNA synthesis began with a lag of about 3 to 5 min after reoxygenation followed by a largely synchronous viral replication round. Viral RNA-DNA primers complementary to the SV40 origin region were not detectable before 3 min upon reoxygenation. A distinct form of circular closed, supercoiled SV40 DNA was detectable as soon as 3 min after reoxygenation but not under hypoxia. Sensitivity to the DNA nucleaseBal 31 and migration behavior in chloroquine-containing agarose gels suggested that this DNA species was highly underwound compared to other SV40 topoisomers and was probably related to the highly underwound form U DNA first described by Dean et al. (F. B. Dean, P. Bullock, Y. Murakami, C. R. Wobbe, L. Weissbach, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 84:16–20, 1987), in vitro. 3′-OH ends of presumed RNA-DNA primers could be detected in form U by 3′ end labeling with T7 polymerase. Addition of aphidicolin to the cells before reoxygenation led to a pronounced accumulation of form U DNA containing RNA-DNA primers. In vivo pulse-chase kinetic studies performed with aphidicolin-treated SV40-infected cells showed that form U is an initial intermediate of SV40 DNA replication which matures into higher-molecular-weight replication intermediates and into SV40 form I DNA after removal of the inhibitor. These results suggest that in vivo initiation of SV40 replication is arrested by hypoxia before origin unwinding and primer synthesis.

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

“…Differential pattern of minichromosome-bound replication proteins occur before and after re-oxygenation of hypoxic cells Initiation of SV40 DNA replication is inhibited at a stage before unwinding in hypoxically cultivated cells. After re-oxygenation, unwinding and RNA-DNA primer synthesis are detectable after 3 min [5].…”

“…To examine whether phosphorylations catalysed by the above mentioned kinases are essential for initiation of SV40 replication after re-oxygenation, we tested the influence of these inhibitors on the formation of SV40 form U, which was shown to be a product of the unwinding of the viral origin region in vivo [5]. The inhibitors were added to the hypoxically cultivated cell cultures 30 min before re-oxygenation.…”

“…Re-oxygenation results in new initiations followed by an almost synchronous round of SV40 replication. This synchronous round of replication was shown to begin at the viral origin [4].After re-oxygenation, the viral replication starts with the unwinding of the viral origin region [5]. This was shown by detection of a highly underwound SV40 topoisomer (form U) about 3 min after re-oxygenation.…”

“…This seems to be of particular significance during embryonic growth, wound healing or tumour cell propagation.The mechanism leading from re-oxygenation to replicon initiation is largely obscure. The remarkably fast resumption of initiations after re-oxygenation suggests that the O 2 -dependent replication control acts very directly on the replication apparatus.O 2 -Dependent regulation of replicon initiation was also demonstrated for viral replication in simian virus 40 (SV40)infected CV1 cells [4,5]. As the replication of SV40 is relatively well investigated, this virus seems to be well suited to examine the events leading to the reversible shutdown of replicon initiations by hypoxia.…”

“…After re-oxygenation, the viral replication starts with the unwinding of the viral origin region [5]. This was shown by detection of a highly underwound SV40 topoisomer (form U) about 3 min after re-oxygenation.…”