HHV-6A was used to infect PHFAs at comparable levels of virus DNA (1 × 108 copies/106 cells) as determined by quantitative PCR. HHV-6A-infected PHFAs showed typical cytopathic effects (CPE) such as cellular swelling and cell fusion at 72 h post-infection (hpi). To further determine HHV-6A infection in PHFAs, the expression of a late protein gp60/110 was analyzed using immunofluorescence assay and western blotting at 72 hpi. As shown in Figure 1b, a prominent expression of HHV-6 gp60/110 was detected in HHV-6A-infected PHFAs compared with that in the control mock-infected cells. The gp60/110 late protein was clearly localized in the cytoplasm of most multinucleate giant cells. Electron microscopic analyses were also performed on HHV-6A-infected PHFAs at 72 hpi. As shown in Figure 1c, viral particles could be visualized in both cytoplasm and extracellular matrix of HHV-6A-infected PHFAs. These results indicate that HHV-6A can cause productive infection in PHFAs.
HHV-6A induces apoptosis of PHFAs
To investigate the effect of HHV-6A infection on apoptosis in PHFAs, cells infected with HHV-6A were stained with annexin-V-FITC and propidium iodide (PI) after 24, 48, and 72 hpi and analyzed by flow cytometry. As shown in Figure 2a, we observed a high percentage of annexin-positive cells (apoptotic cells) in HHV-6A-infected cells at 72 hpi compared to mock-infected cells. The percentage of early apoptotic cells and late apoptotic cells at 72 hpi reached 5.89% and 17.5% compared to 0.64% and 2.48% in mock-infected cells, respectively. To further confirm the effect of HHV-6A on cell apoptosis, we also observed the morphologic changes in HHV-6A-infected cells using transmission electron microscopy. HHV-6A-infected PHFAs showed the typical features of cell apoptosis: marginalized and condensed chromatin matrix, shrinkage and blebbing of the cytoplasm and fragmented nuclei. Virus-like particles could be found in apoptotic HHV-6A-infected PHFAs.
HHV-6A triggers caspases activation
Caspases are synthesized as inactive precursors that are processed to large and small subunits to form the active enzymes. Caspase-3 is one of the main effective caspases, which are activated in response to both intracellular and extracellular death signals. To explore the pathway by which HHV-6A induced apoptosis, we measured caspase-3 activity in HHV-6A-infected PHFAs with anti-active caspase-3 antibody using flow cytometry. PHFAs with activated caspase-3 were about 2.81%, 10.12% and 19.31% at 24, 48 and 72 hpi, respectively, whereas the value was only 0.69% in the mock-infected cells. To further define whether HHV-6A induces apoptosis via the receptor-mediated or the mitochondria-mediated pathways, the activities of caspase-8 and -9 were measured, respectively. HHV-6A infection resulted in significant increases in caspase-8 and caspase-9 activities at 48 and 72 hpi in HHV-6A-infected cells compared with mock-infected cells. These data indicate that HHV-6A induce apoptosis of PHFAs by both the receptor-mediated and the mitochondria-mediated pathways.