Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells.
Coxsackievirus B3 (CVB3), an enterovirus in the family Picornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.
Carthy
C M
CM
Department of Pathology and Laboratory Medicine, University of British Columbia-St. Paul's Hospital, Vancouver, British Columbia, Canada.
Granville
D J
DJ
Watson
K A
KA
Anderson
D R
DR
Wilson
J E
JE
Yang
D
D
Hunt
D W
DW
McManus
B M
BM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3; D020169:Caspases; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003588:Cytopathogenic Effect, Viral; D029822:Enterovirus B, Human / Q000502:physiology*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity
C112287:Ac-aspartyl-glutamyl-valyl-aspartyl-aminomethylcoumarin; D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D011506:Proteins; C106566:benzyloxycarbonyl-aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C109664:caspase-activated DNase inhibitor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases