HHV-6A activates PARP cleavage and up-regulates bax/bcl-2 ratio. Part 3

In addition, NF-κB reportedly induces the expression of c-IAP1, c-IAP2 and XIAP, thereby promoting NF-κB activation in a positive feed-back system. NF-κB up-regulation exerts an anti-apoptotic effect leading to cells survival, transformation, and resistance to radiation and drug therapies. In contrast, NF-κB down-regulation will break this feed-back loop and reduce the expression of c-IAP1, c-IAP2 and XIAP, which are the direct caspase inhibitors. In the present study, we found that HHV-6A decreased NF-κB and increased Iκ-Bα expression in time-dependent manners. IκBα is one member of the family of cellular proteins that function to inhibit the activity of NF-κB. IκBα inhibits NF-κB by masking the nuclear localization signals of NF-κB proteins and keeping them sequestered in an inactive state in the cytoplasm. IκBα up-regulation may inhibit the activity of NF-κB which was observed down-regulation in HHV-6A-infected PHFAs. We also found that HHV-6A decreased expression of c-IAP1, c-IAP2 and XIAP. Increased mitochondrial release of Smac/Diablo could antagonize IAPs expression. Suppression of survival factors such as IAPs and NF-κB could be due to cytosolic up-regulation of Smac/Diablo.


We demonstrated that HHV-6A induces cell apoptosis in PHFAs through both caspase-dependent and -independent apoptosis pathways, as evidenced by (1) activation of caspase-3, -8 and -9; (2) increasing the ratio of Bax/Bcl-2; (3) increasing the presence of Smac/Diablo, AIF and cytochrome c in cytoplasm; (4) down-regulation of anti-apoptotic NF-κB and IAPs. The identification of the apoptotic signaling pathways in HHV-6A-infected PHFAs would be very helpful in understanding the mechanisms by which HHV-6A infection causes diseases in the CNS.

Materials and methods
Cells and viruses

The primary human fetal astrocytes (Sciencell) were cultured in DEME/F12 (Hyclone) supplemented with 10% fetal calf serum, 100 IU/ml penicillin/streptomycin (Invitrogen). Human T-cell line HSB-2 cells were cultured in RPMI 1640 medium containing 8% fetal calf serum. HHV-6A strain GS was inoculated into HSB-2 cells. The cells were frozen and thawed twice when 80% of HHV-6A-infected HSB-2 cells showed the cytopathic effects (CPE), then centrifuged at 2000 × g for 10 min. The supernatants were stored at -70°C as cell-free virus. Viral DNA equivalents of the frozen aliquot were tested by quantitative PCR. Uninfected HSB-2 cells were similarly cultured and treated using the same procedure and used for mock infection. For infection, 3 × 105 primary human fetal astrocytes (PHFAs) were seeded onto a poly-L-lysine (Sigma)-coated 25-cm2 flask (Corning). After an overnight incubation, the plate was washed three times with phosphate-buffered saline (PBS) and infected with cell-free supernatant containing 108 viral DNA copies/106 PHFAs. After 3 h incubation at 37°C in 5% CO2, cultures were washed three times with PBS, and fresh medium was added. The HHV-6A-infected cells were checked for CPE every day in microscopy. Procedures for mock infection were performed in the same manner as for viral infection.