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Ectromelia virus RING finger protein is localized in virus factories and is required for virus replication in
T G Senkevich1, E J Wolffe, R M Buller
1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
Abstract:
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.
Insights
Ectromelia virus (EV) protein p28 is essential for viral replication in macrophages, unlike in cell cultures. This protein is crucial for EV pathogenicity by enabling replication in these specific immune cells.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- A previously identified ectromelia virus (EV) gene encodes a 28-kDa RING zinc finger protein (p28).
- p28 is nonessential for EV growth in cell culture but critical for pathogenicity in mice.
- EV pathogenesis involves complex interactions with host immune cells.
Purpose of the Study:
- To investigate the role of p28 in EV replication within macrophages.
- To elucidate the mechanism by which p28 influences viral DNA replication.
- To understand the contribution of p28 to EV pathogenicity in vivo.
Main Methods:
- In vitro replication assays using EV and p28- mutant in murine resident peritoneal macrophages.
- Analysis of viral DNA replication and early protein synthesis.
- Immunofluorescence and biochemical analyses to determine p28 localization.
- Vaccinia virus expression system to study truncated p28 variants.
- Inhibition of viral DNA replication using cytosine arabinoside.
Main Results:
- p28 expression is required for EV in vitro replication in macrophages, but not in other cell cultures.
- EV p28- mutant showed no viral DNA replication in macrophages, despite early protein synthesis.
- p28 localizes to virus factories in infected macrophages and BSC-1 cells.
- Disruption of the RING domain did not affect p28 intracellular localization.
- p28 formed distinct focal structures when viral DNA replication was inhibited.
Conclusions:
- p28 likely substitutes for unknown cellular factors required for EV DNA replication or a later stage of viral reproduction in macrophages.
- The requirement for p28 in macrophages explains the attenuated pathogenicity of p28- mutants.
- EV replication failure in macrophages due to p28 absence impacts virus spread and liver targeting.