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 injur es multiple susceptible organs and tissues in vivo, including the myoc ardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa prefo rm of caspase 3 is cleaved subsequent to the degenerative morphologica l changes seen in infected HeLa cells. Caspase activation assays confi rm 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 respo nsible for DNA fragmentation, were degraded at 9 h following infection , yielding their characteristic cleavage fragments. Inhibition of casp ase activation by benzyloqcarbonyl-Val-Ala-Asp-fluoromethylketone (ZVA D.fmk) did not inhibit the virus-induced cytopathic effect, while inhi bition of caspase activation by ZVAD.fmk in control apoptotic cells in duced by treatment with the porphyrin photosensitizer benzoporphyrin d erivative monoacid ring A and visible light inhibited the apoptotic ph enotype. Caspase activation and cleavage of substrates may not be resp onsible for the characteristic cytopathic effect produced by picornavi rus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.