Novel vectors expressing anti-apoptotic protein Bcl-2 to study cell death in Semliki Forest virus-infected cells.
Semliki Forest virus (SFV, Alphavirus) induce rapid shut down of host cell protein synthesis and apoptotic death of infected vertebrate cells. Data on alphavirus-induced apoptosis are controversial. In this study, the anti-apoptotic bcl-2 gene was placed under the control of duplicated subgenomic promoter or different internal ribosome entry sites (IRES) and expressed using a novel bicistronic SFV vector. The use of IRES containing vectors resulted in high-level Bcl-2 synthesis during the early stages of infection. Nevertheless, in infected BHK-21 cells translational shutdown was almost complete by 6h post-infection, which was similar to infection with appropriate control vectors. These results indicate that very early and high-level bcl-2 expression did not have a protective effect against SFV induced shutdown of host cell translation. No apoptotic cells were detected at those time points for any SFV vectors. Furthermore, Bcl-2 expression did not protect BHK-21 or AT3-neo cells at later time points, and infection of BHK-21 or AT3-neo cells with SFV replicon vectors or with wild-type SFV4 did not lead to release of cytochrome c from mitochondria. Taken together, our data suggest that SFV induced death in BHK-21 or AT3-neo cells is not triggered by the intrinsic pathway of apoptosis.
Kiiver
Kaja
K
Estonian Biocentre, Riia Street 23, 51010 Tartu, Estonia.
Merits
Andres
A
Sarand
Inga
I
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D002478:Cells, Cultured; D015967:Gene Expression Regulation, Viral; D019254:Genes, bcl-2 / Q000235:genetics / Q000502:physiology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D012672:Semliki forest virus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*
D049452:Green Fluorescent Proteins