Ectromelia virus RING finger protein is localized in virus factories and is required for virus replication in macrophages.

We have previously described a gene of ectromelia virus (EV) that codes for a 28-kDa RING zinc finger-containing protein (p28) that is nonessential for virus growth in cell culture but is critical for EV pathogenicity in mice (T. G. Senkevich, E. V. Koonin, and R. M. L. Buller, Virology 198:118-128; 1994). Here, we show that, unlike all tested cell cultures, the expression of p28 is required for in vitro replication of EV in murine resident peritoneal macrophages. In macrophages infected with the p28- mutant, viral DNA replication was not detected, whereas the synthesis of at least two early proteins was observed. Immunofluorescence and biochemical analyses showed that in EV-infected macrophages or BSC-1 cells, p28 is associated with virus factories. By use of a vaccinia virus expression system to examine different truncated versions of p28, it was shown that the disruption of the specific structure of the RING domain had no influence on the intracellular localization of this protein. When viral DNA replication was inhibited with cytosine arabinoside, p28 was found in distinct, focal structures that may be precursors to the factories. We hypothesize that in macrophages, which are highly specialized, nondividing cells, p28 substitutes for an unknown cellular factor(s) that may be required for viral DNA replication or a stage of virus reproduction between the expression of early genes and the onset of DNA synthesis. In the absence of p28, the attenuation of EV pathogenicity can be explained by a failure of the virus to replicate in macrophage lineage cells at all successive steps in the spread of virus from the skin to its target organ, the liver.