Replication of African swine fever virus DNA in infected cells

We have examined the ultrastructural localization of African swine fever vi rus DNA in thin-sections of infected cells by in situ hybridization and aut oradiography. Virus-specific DNA sequences were found in the nucleus of inf ected 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 th at the nucleus is also a site of viral DNA replication at early times. Thes e results provide further evidence of the existence of nuclear and cytoplas mic stages in the synthesis of African swine fever virus DNA. On the other hand, alkaline sucrose sedimentation analysis of the replicative intermedia tes synthesized in the nucleus and cytoplasm of infected macrophages showed that small DNA fragments (similar to 6-12S) were synthesized in the nucleu s at an early time, whereas at later times, larger fragments of similar to 37-49S were labeled in the cytoplasm. Pulse-chase experiments demonstrated that these fragments are precursors of the mature cross-linked viral DNA. T he formation of dimeric concatemers, which are predominantly head-to-head l inked, 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 swin e fever virus DNA proceeds by a de novo start 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-u nit concatemer with dimeric ends that could be resolved to generate the gen omic DNA by site-specific nicking, rearrangement, and ligation as has been proposed in the de novo start model of Baroudy at al. (B. M. Baroudy, S. Ve nkatesam, and B. Moss, 1982, Cold Spring Harbor Symp. Quant. Biol. 47 723-7 29) for the replication of vaccinia virus DNA. (C) 1999 Academic Press.