Replication of African swine fever virus DNA in infected cells

We have examined the ultrastructural localization of African swine fever virus DNA in thin-sections of infected cells byin situhybridization and autoradiography. Virus-specific DNA sequences were found in the nucleus of infected Vero cells at early times in the synthesis of the viral DNA, forming dense foci localized in proximity to the nuclear membrane. At later times, the viral DNA was found exclusively in the cytoplasm. Electron microscopic autoradiography of African swine fever virus-infected macrophages showed that the nucleus is also a site of viral DNA replication at early times. These results provide further evidence of the existence of nuclear and cytoplasmic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermediates synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments (∼6–12S) were synthesized in the nucleus at an early time, whereas at later times, larger fragments of ∼37–49S were labeled in the cytoplasm. Pulse–chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. The formation of dimeric concatemers, which are predominantly head-to-head linked, was observed by pulsed-field electrophoresis and restriction enzyme analysis at intermediate and late times in the replication of African swine fever virus DNA. Our findings suggest that the replication of African swine fever virus DNA proceeds by ade novostart mechanism with the synthesis of small DNA fragments, which are then converted into larger size molecules. Ligation or further elongation of these molecules would originate a two-unit concatemer with dimeric ends that could be resolved to generate the genomic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in thede novostart model of Baroudyet al.(B. M. Baroudy, S. Venkatesam, and B. Moss, 1982,Cold Spring Harbor Symp. Quant. Biol.47, 723–729) for the replication of vaccinia virus DNA.This work was supported by grants from the Direccio´ n General de Investigacio´ n Cientı´fica y Te´ cnica (PB96–0902-C02–01 and AGF98–1352-CE) and the European Community (FAIR5-PL97–3441) and an institutional grant from the Fundacio´ n Ramo´ n Areces to the Centro de Biologı´a Molecular ‘‘Severo Ochoa.’