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bcl-2 acts early to restrict Semliki Forest virus replication and delays virus-induced programmed cell death
M F Scallan1, T E Allsopp, J K Fazakerley
1Department of Veterinary Pathology, University of Edinburgh, United Kingdom.
Abstract:
As characterized by morphological assessment and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, Semliki Forest virus (SFV) infection of rat prostatic adenocarcinoma cells triggers an apoptotic cell response. Cell death proceeded more rapidly following infection with the neurovirulent L10 strain of SFV than with the avirulent A7 strain. Overexpression of the antiapoptotic proto-oncogene bcl-2 allowed survival of cultures infected with either strain of virus. bcl-2 overexpression drastically reduced the numbers of productively infected cells within the cultures. In situ hybridization for viral message-sense RNA coupled with immunostaining for viral protein indicated that bcl-2 functions at an early stage of the virus life cycle, at entry, pretranscriptional events or at transcription, to inhibit virus replication. Double-immunofluorescent labeling for bcl-2 and viral glycoproteins revealed double-positive cells, demonstrating that with time, this early block in replication can be overcome. These productively infected bcl-2-expressing cells do, with time, undergo apoptosis. As a result of changing the balance between cell death and cell division by restricting productive virus replication and delaying virus-induced cell death, bcl-2 expression led to the establishment of chronically infected cell lines which could be passaged.
Insights
Semliki Forest virus (SFV) infection induces apoptosis in cancer cells. Overexpressing bcl-2 protein inhibits SFV replication and cell death, leading to chronic infections.
Area of Science:
- Virology
- Cell Biology
- Cancer Research
Background:
- Semliki Forest virus (SFV) infection in rat prostatic adenocarcinoma cells induces apoptosis.
- Apoptosis occurs faster with the neurovirulent L10 SFV strain compared to the avirulent A7 strain.
Purpose of the Study:
- To investigate the role of the antiapoptotic proto-oncogene bcl-2 in SFV-induced apoptosis.
- To determine the effect of bcl-2 on SFV replication and the establishment of chronic infections.
Main Methods:
- Morphological assessment and TUNEL assay for apoptosis.
- Overexpression of bcl-2 in infected cells.
- In situ hybridization for viral RNA and immunostaining for viral protein.
- Double-immunofluorescent labeling for bcl-2 and viral glycoproteins.
Main Results:
- bcl-2 overexpression allowed cell survival and drastically reduced productive infection.
- bcl-2 inhibited SFV replication at an early stage (entry, pretranscriptional events, or transcription).
- The early replication block was overcome over time, leading to apoptosis in bcl-2-expressing cells.
- bcl-2 expression led to the establishment of chronically infected, passaged cell lines.
Conclusions:
- bcl-2 plays a critical role in regulating SFV-induced apoptosis and viral replication.
- Modulating the balance between cell death and division via bcl-2 can establish persistent viral infections.