Hypoxia blocks in vivo initiation of simian virus 40 replication at a stage preceding origin unwinding

Simian virus 40 (SV40)-infected CV1 cells transiently exposed to hypoxia sh ow a burst of viral replication immediately after reoxygenation, DNA precur sor incorporation and analysis of growing daughter strands by alkaline sedi mentation demonstrated that SV40 DNA synthesis began with a lag of about 3 to 5 min after reoxygenation followed by a largely synchronous viral replic ation round. Viral RNA-DNA primers complementary to the SV40 origin region were not detectable before 3 min upon reoxygenation, A distinct form of cir cular closed, supercoiled SV40 DNA was detectable as soon as 3 min after re oxygenation but not under hypoxia. Sensitivity to the DNA nuclease Bal 31 a nd 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 describe d by Dean et al. (F. B. Dean, P. Bullock, Y. Murakami, C. R, Wobbe, L. Weis sbach, 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 befor e reoxygenation led to a pronounced accumulation of form U DNA containing R NA-DNA primers. In vivo pulse-chase kinetic studies performed with aphidico lin-treated SV40-infected cells showed that form U is an initial intermedia te of SV40 DNA replication which matures into higher-molecular-weight repli cation intermediates and into SV40 form I DNA after removal of the inhibito r. These results suggest that in vivo initiation of SV40 replication is arr ested by hypoxia before origin unwinding and primer synthesis.