HHV-6 was first isolated from peripheral blood mononuclear cells of patients with lymphoproliferative disorders and AIDS. There are two variants of HHV-6 (A and B) according to distinct genetic, immunological and virological characteristics. As with other virus, HHV-6 is able to induce apoptosis of host cells. Subsequent studies have demonstrated that HHV-6 has been shown to induce apoptosis in astrocytes, oligodendrocytes, neuronal cell lines and CD4+ T lymphocytes. Gardell et al. reported that HHV-6A induced apoptosis by an unknown mechanism in astrocytes, oligodendrocytes and neuronal cell lines. Inoue et al. demonstrated that TNF-α and anti-Fas antibodies augmented HHV-6-induced apoptosis, suggesting an involvement bof death receptors in HHV-6-induced apoptosis. In contrast, Inchimi et al. found that HHV-6 induced apoptosis of cord blood lymphocytes through a receptor-independent pathway.
Caspases play a critical role in apoptosis, which cleave specific substrates and activate downstream molecules and culminate in cell death. However, the roles of caspases in HHV-6-induced apoptosis of astrocytes haven’t been studied yet. In this study, we demonstrated that the activities of caspase-3, -8 and -9 were all increased in HHV-6A-induced apoptosis of PHFAs. In addition, we found that PARP was cleaved in HHV-6A-induced apoptotic PHFAs. Caspase-3 is a common effector of both death receptor and the mitochondrial signaling pathways. Caspase-8 is activated by the death receptor signaling pathway, whereas caspase-9 is activated in the mitochondrial signaling pathway during apoptosis. We speculate that HHV-6A-induced apoptosis in astrocytes via both caspase-dependent receptor and mitochondrial apoptotic pathways.
Bcl-2 family proteins are central regulators of the mitochondrial apoptotic pathway and have been implicated in various models of virus-induced apoptosis. Pugazhenthi et al. found that simian varicella virus induced apoptosis in monkey kidney cells via caspase-dependent mitochondrial pathway and involves down-regulation of bcl-2 expression. The translocation and accumulation of Bax, a pro-apoptotic factor of Bcl-2 family in mitochondria will lead to release of cytochrome c and AIF. We examined the expression of Bcl-2 and Bax in HHV-6A-induced mitochondrial dysfunction. Our data showed that the anti-apoptotic protein Bcl-2 decreased, which was accompanied by the increase of pro-apoptotic protein Bax during HHV-6A infection, suggesting that Bcl-2 and Bax were involved in the apoptosis of HHV-6A-infected PHFAs.
Up-regulation of Bax induces the permeabilization of mitochondrial outer membrane and initiates mitochondrial dysfunction. Mitochondria may release several molecules including cytochrome c, Smac/Diablo, and AIF to induce apoptosis via the caspase-dependent and -independent pathways [24-26]. We separated the cytosolic and mitochondrial fractions to examine the cytochrome c levels by Western blotting and found a marked increase in cytochrome c level in the cytosolic fraction due to a concomitant decrease in the cytochrome c level in the mitochondrial fraction following HHV-6A infection. Mitochondrial cytochrome c releases into cytoplasm to bind to the apoptosis protease activation factor (APAF1) and to form a complex of apoptosome activating pro-caspase 9. The activation of pro-caspase 9 initiates an enzymatic reaction cascade leading to the execution of apoptosis in cells. We also observed a time-dependent increase the cytosolic level of Smac/Diablo following infection compared with control cells. Smac/Diablo can inhibit inhibitor-of-apoptosis-proteins (IAPs), which otherwise inactivate caspases. Mitochondria-mediated apoptosis may also occur caspase-independently after mitochondrial release of AIF and Endo G which are translocated to the nucleus for induction of chromatin condensation and DNA fragmentation. Our investigation demonstrated that HHV-6A markedly increased the cytosolic level of AIF in PHFAs, indicating involvement of caspase-independent pathway of apoptosis.