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 code s for a 28-kDa RING zinc finger-containing protein (p28) that is nones sential for virus gro,vth in cell culture but is critical for EV patho genicity 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 cel l cultures, the expression of p28 is required for in vitro replication of EV in murine resident peritoneal macrophages. In macrophages infec ted with the p28(-) mutant, viral DNA replication was not detected, wh ereas the synthesis of at least two early proteins was observed. Immun ofluorescence and biochemical analyses showed that in EV-infected macr ophages 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 stru cture of the RING domain had no influence on the intracellular localiz ation of this protein. When viral DNA replication was inhibited with c ytosine 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 repl ication or a stage of virus reproduction between the expression of ear ly genes and the onset of DNA synthesis. In the absence of p28, the at tenuation of EV pathogenicity can be explained by a failure of the vir us to replicate in macrophage lineage cells at all successive steps in the spread of virus from the skin to its target organ, the liver.