Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2.
Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.
Grandgirard
D
D
Institute of Medical Microbiology, University of Berne, Friedbuehlstrasse 51, CH-3010 Berne, Switzerland.
Studer
E
E
Monney
L
L
Belser
T
T
Fellay
I
I
Borner
C
C
Michel
M R
MR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D000939:Epitopes / Q000032:analysis; D005347:Fibroblasts; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012672:Semliki forest virus / Q000502:physiology*; D012845:Sindbis Virus / Q000502:physiology*; D051028:bcl-2-Associated X Protein
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D000939:Epitopes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases