Poxvirus DNA. II. Replication of vaccinia virus DNA in the cytoplasm of HeLa cells.

Abstract

Virions, purified by Method I (containing genomes which lack cross-links and appear fragmented when analyzed in alkaline sucrose gradients) or by Method II (containing cross-linked genomes which sedimented at ∼ 102 S in alkaline sucrose gradients with no evidence of fragmentation) were found to be equally efficient in replicating their DNA in the cytoplasm of HeLa cells. Replication of vaccinia DNA in the cytoplasm of infected cells was discontinuous; that is, short pulses of [3H]thymidine were incorporated into fragments that sedimented at about 20 S in alkaline sucrose. As the pulse length increased, label was found in material which sedimented faster than 20 S but more slowly than the strands of mature viral DNA. Molecules sedimenting at 30–40 S in alkaline sucrose appeared to accumulate with longer pulses. During a “chase” in cold medium, part of these molecules were converted to full-length molecules sedimenting at 72–75 S and into mature, cross-linked genomes sedimenting at 102–106 S in alkaline sucrose. Chromatography on BND-cellulose and hydroxyapatite columns demonstrated that replicating viral DNA had single-stranded regions, but such molecules could not be separated from mature genomes by sedimentation in CsCl gradients. No nascent strands of greater than unit length were detected indicating that replication of the vaccinia genome probably does not involve continuous concatemers or the addition of new material to parental strands. This suggests that polymerization may be symmetrical. Evidence for a circular intermediate, with nicks in both strands, was obtained by the analysis of “pulse” and “pulsechase” labeled replicating viral DNA molecules by centrifugation in neutral sucrose gradients. DNA species sedimenting 12–15% more rapidly than parental genomes, at 82–86 S, appropriate for such a “nicked” circular intermediate were found, in which label accumulated during a chase before the appearance of mature genomes at 68–72 S.