Bcl-2 blocks a caspase-dependent pathway of apoptosis activated by herpes simplex virus 1 infection in HEp-2 cells

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants indu ce programmed cell death and that wild-type virus blocks the execution of t he cell death program triggered by expression of viral genes, by the Fas an d tumor necrosis factor pathways, or by nonspecific stress agents. In parti cular, an earlier report from this laboratory showed that the mutant virus d120 lacking the genes encoding infected cell protein 4 (ICP4), the major r egulatory protein of the virus, induces a caspase-3-independent pathway of apoptosis in human SK-N-SH cells. Here we report that the pathway of apopto sis induced by the d120 mutant in human HEp-2 cells is caspase dependent. S pecifically, in HEp-2 cells infected with d120, (i) a broad-range inhibitor of caspase activity, z-vad-FMK, efficiently blocked DNA fragmentation, (ii ) cytochrome c was released into the cytoplasm, (iii) caspase-3 was activat ed inasmuch as poly(ADP-ribose) polymerase was cleaved, and (iv) chromatin condensation and fragmentation of cellular DNA were observed. In parallel s tudies, HEp-2 cells were transfected with a plasmid encoding human Bcl-2 an d a clone (VAX-3) expressing high levels of Bcl-2 was selected. This report shows that Bcl-2 blocked all of the manifestations associated with program med cell death caused by infection with the d120 mutant. Consistent with th eir resistance to programmed cell death, VAX-3 cells overproduced infected cell protein 0 (ICP0). An unexpected observation was that ICP0 encoded by t he d120 mutant accumulated late in infection in small, quasi-uniform vesicl e-like structures in all cell lines tested. Immunofluorescence-based coloca lization studies indicated that these structures were not mitochondria or c omponents of the endoplasmic reticulum or the late endosomal compartment. T hese studies affirm the conclusion that HSV can induce programmed cell deat h at multiple steps in the course of its replication, that the d120 mutant can induce both caspase-dependent and -independent pathways of programmed c ell death, and that virus-induced stimuli of programmed cell death may diff er with respect to the pathway that they activate.