Summary auto-generated
This study demonstrates that Semliki Forest virus (SFV), an enveloped RNA virus of the alphavirus genus, induces programmed cell death (apoptosis) in cultured cells through a mechanism independent of the p53 tumor suppressor gene. Researchers transfected baby hamster kidney (BHK) cells and human lung carcinoma cells with RNA from wild-type SFV and various deletion mutants. DNA laddering and TUNEL staining confirmed apoptosis in cells expressing viral nonstructural proteins when viral RNA replication occurred. Critically, apoptosis was induced even in H358a cells that completely lack p53 expression due to homozygous deletion. However, a deletion mutant lacking the nsP2 nonstructural gene—required for viral RNA synthesis—failed to induce apoptosis or produce viral RNA. The SFV1 expression vector, which lacks structural protein genes but retains nonstructural genes, similarly induced p53-independent apoptosis. These findings indicate that an apoptosis-inducing function resides in the nonstructural coding region of SFV and depends critically on viral RNA synthesis capability. The results suggest double-stranded RNA synthesis associated with viral replication may be the trigger, linking previously recognized cytopathogenic effects to apoptotic mechanisms.
Key findings
- SFV nonstructural proteins induce apoptosis independent of p53 status, demonstrated by apoptosis induction in H358a cells lacking functional p53
- Viral RNA synthesis is essential for apoptosis induction, as the nsP2 deletion mutant (lacking viral replication capability) fails to trigger apoptosis
- The SFV1 expression vector, containing only nonstructural genes with a multicloning site replacing structural genes, induces apoptosis comparable to wild-type virus
- Apoptosis was detected by DNA laddering and TUNEL staining 48 hours after transfection in multiple cell types
- Double-stranded RNA synthesis during viral replication is likely the mechanism triggering p53-independent apoptosis
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Abstract
Three deletion mutants of the structural protein region of the Semliki Forest virus (SFV) genome, including one which encompassed all the viral structural protein genes, induced apoptosis in BHK cells at 48 h after transfection, as shown by DNA laddering and TUNEL staining, as did the wild-type SFV4 RNA. A similar result was obtained for the SFV1 expression vector, which has a multicloning site inserted in place of the structural protein genes. However, in cells transfected with viral RNA containing a deletion of the nsP2 gene, neither viral RNA synthesis nor the induction of apoptosis occurred. Both SFV1 vector and wild-type SFV4 RNA induced apoptosis in human H358a lung carcinoma cells, which have a homozygous deletion of the p53 gene. It is concluded that the SFV vector encodes a function in the nonstructural coding region which induces p53-independent apoptosis and is dependent on viral RNA synthesis.