Human herpesvirus 6A induces apoptosis of primary human fetal astrocytes via both caspase-dependent and -independent pathways

Abstract

Background: Human herpesvirus 6 (HHV-6) is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS) diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS) and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs) during productive HHV-6A infection and the underlying mechanisms.

Results: HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP), which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs.

Conclusion: This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.

Figure 1

HHV-6A causes infection in PHFAs. a. HHV-6A infection exhibited typical cytopathic effects in infected PHFAs. The morphological characteristics of PHFAs infected with or without HHV-6A were observed under light microscope. b. HHV-6A-infected PHFAs express viral gp60/110 protein at 72 h post-infeciton. The gp60/110 protein was determined by IFA and western blotting with an anti-gp60/110 monoclonal antibody. c. Electron microscopic photographs of typical herpesvirus-like particles were observed in both cytoplasm and extracellular matrix of HHV-6A-infected PHFAs.

Figure 2

HHV-6A infection induces apoptosis of PHFAs. a. Mock- and HHV-6A-infected PHFAs were stained with annexin V-PI and analyzed by flow cytometry. Percentage of apoptotic cells was summarized. Each column represents the mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001). b. Electron microscopic photographs of mock- and HHV-6A-infected PHFAs. c. Electron microscopic photographs of virus-like particles in apoptotic HHV-6A-infected PHFAs.

Figure 3

HHV-6A triggers caspases activation. a. Mock- and HHV-6A-infected PHFAs were collected at various time points and the levels of activated caspase-3 were measured by flow cytometry. b-c. The activation of caspase-8 and caspase-9 was examined by colorimetric method using lysates from mock-infected and HHV-6A-infected PHFAs. Each column represents the mean ± SD of three independent experiments (***P < 0.001).

Figure 4

HHV-6A activates PARP cleavage and up-regulates Bax/Bcl-2 ratio. a. PARP in mock-infected and HHV-6A-infected cells was analyzed by Western blotting. b. Expressions of Bcl-2 and Bax were detected by Western blots using anti-Bcl-2 and anti-Bax antibodies, respectively. β-actin was used as a loading control. Quantitative values of Bcl-2 and Bax are the mean ± SD from three independent experiments (**P < 0.01, ***P < 0.001).

Figure 5

HHV-6A infection results in the release of pro-apoptotic proteins from mitochondria. Expressions of pro-apoptotic proteins liberated from mitochondria were detected by Western blots as described in Methods and Materials. β-actin was used as a loading control.

Figure 6

Down-regulation of anti-apoptotic proteins by HHV-6A infection. Representative Western blots show levels of expression of NF-κB, IκBα, c-IAP1, c-IAP2 and XIAP. β-actin was used as a loading control.