HHV-6A activates PARP cleavage and up-regulates bax/bcl-2 ratio

PARP is an established substrate for caspase-3 in the apoptotic events. Cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis. Western blotting was used to detect endogenous full-length PARP (116 KD), as well as the large fragment (89 KD) of PARP resulting from caspase cleavage. As shown in Figure 4a, the 89 KD cleaved fragment of PARP was detected in infected cells at 48 and 72 hpi, but not detected in the mock-infected cells.

The mitochondria-mediated pathway of apoptosis is regulated by the Bcl-2 family proteins, which are known to directly regulate mitochondrial membrane permeability. We examined the levels of expression of Bax (pro-apoptotic) and Bcl-2 (anti-apoptotic) proteins using Western blotting analysis. As shown in Figure 4b, the levels of Bcl-2 protein were significantly decreased following HHV-6A infection compared to that in mock-infected cells, whereas the expression of Bax protein was significantly increased in HHV-6A-infected cells. These results indicate Bax/Bcl-2 ratio was significantly increased in HHV-6A-infected cells compared with mock-infected cells.

HHV-6A infection results in the release of pro-apoptotic proteins from mitochondria

Mitochondria may release several molecules including cytochrome c, Smac/Diablo, and AIF to induce apoptosis. Mitochondrial cytochrome c release is a well-known pre-condition for formation of apoptosome and activation of caspases for apoptosis. There is a marked increase in the levels of cytochrome c released from mitochondria to cytoplasm at 48 and 72 hpi compared with control. Smac/Diablo plays an important role in apoptosis by down-regulation anti-apoptotic IAPs. The expression levels of Smac/Diablo were significantly increased following HHV-6A infection in a time-dependent manner compared to that in mock-infected cells. In addition, the expression levels of AIF, determining caspase-independent pathway of apoptosis were also increased obviously in HHV-6A infected cells compared to that in mock-infected cells. The results suggest that HHV-6A infection in PHFAs can provoke apoptosis via the mitochondrial intrinsic pathway.

HHV-6A suppresses IAPs and NF-κB-mediated anti-apoptosis effect

IAPs are thought to function primarily by negative regulation caspases, which are cysteine proteases involved in apoptosis. In human cells, IAPs mainly include cIAP1, cIAP2 and XIAP. As shown in Figure 6, the levels of these three IAPs were significantly decreased in the HHV-6A-infected cells compared to those in mock-infected cells.

NF-κB plays a crucial role not only in immunity, inflammation and cell migration but also in cell survival and apoptosis. Many studies confirmed that NF-κB up-regulation and activation exerted an anti-apoptotic effect leading to cells survival, transformation, and resistance to radiation and chemotherapy. The levels of NF-κB were significantly decreased in HHV-6A-infected cells compared to that in mock-infected cells, while the expression of NF-κB inhibitor-IκBα protein was significantly increased in HHV-6A-infected cells. These results indicate that HHV-6A infection injures IAPs and NF-κB-mediated anti-apoptosis signal pathways in PHFAs.