Related Experiment Videos
Ectromelia virus virulence factor p28 acts upstream of caspase-3 in response to UV light-induced apoptosis
D J Brick1, R D Burke, A A Minkley
1Departments of Biochemistry and Microbiology and Biology, University of Victoria, PO Box 3055, Victoria, BC, Canada V8W 3P6.
Abstract:
Ectromelia virus (EV) virulence factor p28 (EVp28) is a member of a family of poxvirus proteins that are defined largely by the presence of a C-terminal RING finger motif and localization to virus factories within the cytoplasm of infected cells. Previously, overexpression of the Shope fibroma virus (SFV) homologue, N1R, in vaccinia virus (VV)-infected BGMK cells was found to inhibit virus-induced apoptosis. Here, we report that both EVp28 and overexpression of SFV N1R in poxvirus-infected HeLa cells protect specifically from UV light-induced apoptosis, but not from apoptosis induced by Fas or TNF. Further, we report that both VV and EV protect from apoptosis induced by UV, Fas and TNF. Immunoblot analysis indicates that EVp28 acts upstream of caspase-3, blocking activation of the protease in response to UV irradiation. Although no difference was found in replication of an EVp28(-) mutant virus, which expresses a truncated p28 protein lacking the RING motif, compared to EV wild-type in HeLa cells, UV irradiation of infected HeLa cells reduced the replication of the EV mutant compared with wild-type EV.
Insights
Ectromelia virus p28 (EVp28) and Shope fibroma virus N1R protect cells from UV-induced apoptosis by inhibiting caspase-3 activation. This suggests a role for poxvirus proteins in modulating cell death pathways.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Poxvirus proteins, including Ectromelia virus p28 (EVp28), possess a C-terminal RING finger motif and localize to cytoplasmic virus factories.
- Overexpression of Shope fibroma virus (SFV) N1R has previously been shown to inhibit virus-induced apoptosis in vaccinia virus (VV)-infected cells.
Purpose of the Study:
- To investigate the role of EVp28 and SFV N1R in apoptosis induced by different stimuli.
- To determine the mechanism by which EVp28 affects apoptosis and viral replication.
Main Methods:
- HeLa cells were infected with poxviruses, including EV and VV.
- Apoptosis was induced using UV light, Fas, or TNF.
- Immunoblot analysis was used to assess caspase-3 activation.
- Viral replication was compared between wild-type EV and an EVp28 mutant virus.
Main Results:
- Both EVp28 and SFV N1R specifically protected poxvirus-infected HeLa cells from UV-induced apoptosis, but not from Fas or TNF-induced apoptosis.
- VV and EV exhibited protection against UV, Fas, and TNF-induced apoptosis.
- EVp28 acted upstream of caspase-3, preventing its activation following UV irradiation.
- UV irradiation reduced the replication of an EVp28 mutant virus lacking the RING motif compared to wild-type EV.
Conclusions:
- EVp28 and SFV N1R are involved in protecting poxvirus-infected cells from UV-induced apoptosis.
- EVp28 functions by inhibiting caspase-3 activation.
- The p28 RING motif is important for efficient viral replication under UV stress.