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

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

Virology 72(1): 134-146

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 .apprx. 102S in alkaline sucrose gradients with no evidence of fragmentation) were equally in replicating their DNA in the cytoplasm of HeLa cells. Replication of vaccinia DNA in the cytoplasm of infected cells was discontinuous; i.e., short pulses of [3H]thymidine were incorporated into fragments that sedimented at about 20S in alkaline sucrose. As the pulse length increased, label was found in material which sedimented faster than 20S but more slowly than the strands of mature viral DNA. Molecules sedimenting at 30-40S 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 72-75S and into mature, cross-linked genomes sedimenting at 102-106S in alkaline sucrose. Chromatography on BND [benzoylated napthoylated DEAE] 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 additon of new material to parental strands. Polymerization may be symmetrical. Evidence for a circular intermediate, with nicks in both strands, was obtained by the analysis of pulse and pulse-chase labeled replicating viral DNA molecules by centrifugation in neutral sucrose gradients. DNA species sedimenting 12-15% more rapidly than parental genomes, at 82-86S, appropriate for such a nicked circular intermediate were found, in which label accumulated during a chase before the appearance of mature genomes at 68-72S.

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Accession: 068538433

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PMID: 936479

DOI: 10.1016/0042-6822(76)90318-4

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