Classical swine fever virus infection protects aortic endothelial cells from pIpC-mediated apoptosis

Abstract

Classical swine fever virus (CSFV) causes severe disease in pigs associated with leukopenia, haemorrhage and fever. We show that CSFV infection protects endothelial cells from apoptosis induced by the dsRNA mimic, pIpC, but not from other apoptotic stimuli, FasL or staurosporine. CSFV infection inhibits pIpC-induced caspase activation, mitochondrial membrane potential loss and cytochrome c release as well as the pro-apoptotic effects of truncated Bid (tBid) overexpression. The CSFV proteins N pro and E rns both contribute to CSFV inhibition of apoptosis. We conclude that CSFV infection can inhibit apoptotic signalling at multiple levels, including at the caspase-8 and the mitochondrial checkpoints. By supporting viral replication, endothelial cells may promote CSFV pathogenesis. INTRODUCTIONClassical swine fever virus (CSFV), a pestivirus within the family Flaviviridae, is related in terms of its sequence and genome organization to other members of the flavivirus group that give rise to human diseases such as hepatitis C and dengue fever. CSFV is a positive-sense RNA virus, and is the causative agent of classical swine fever (CSF), a notifiable disease of pigs (Le Potier et al., 2006). Disease outbreaks are associated with considerable economic loss and adverse effects on animal welfare (Paton & GreiserWilke, 2003). In its most virulent form, CSFV infection causes severe immunopathological signs, including leukopenia, haemorrhage and fever, accompanied by high morbidity and mortality. However, within an infected population, some animals are found to recover or develop chronic disease (Le Potier et al., 2006). These observations suggest a complex interplay between the virus and the host immune system. CSFV infection is accompanied by depression of cellular immune defences (Ganges et al., 2008) and particularly innate responses mediated by interferon (Bensaude et al., 2004; La Rocca et al., 2005; Ruggli et al., 2005;Seago et al., 2007). In vivo, elevated levels of cell death by apoptosis are thought to account for the characteristic depletion of circulating leukocytes (Sanchez-Cordon et al., 2002;Summerfield et al., 2000Summerfield et al., , 2001. However, it has been found that apoptotic cells rarely contain detectable viral antigen, indicating that apoptotic cells are generally not CSFV-infected (Choi et al., 2004;Sato et al., 2000;Summerfield et al., 2001). This finding, together with the observation that acutely infected animals develop high viral loads, suggest a possible role for cell populations that are resistant to virus-induced apoptosis. These may include endothelial cells.Apoptosis is a controlled cell death programme employed by multicellular organisms to eliminate damaged, aberrant or infected cells (Benedict et al., 2002). A characteristic of apoptotic cell death is the activation of the caspase family of proteases. Caspases that function near the apex of cell death cascades are designated initiators and include caspases-8 and -9, whereas those involved in the terminal stages of cell death, such as ...

The classical swine fever virus N-terminal protease Npro binds to cellular HAX-1

“…We suggest that it is feasible that, to ensure efficient viral replication, the CSFV N pro product targets the apoptotic response through its interaction with HAX-1. CSFV infection inhibits dsRNA-induced apoptosis at a number of apoptotic checkpoints, preventing caspase-8 activation and loss of mitochondrial membrane potential (Johns et al, 2010). N pro contributes to the antagonism of dsRNA-mediated apoptosis; however the underlying mechanism has not been determined (Johns et al, 2010;Ruggli et al, 2003Ruggli et al, , 2005.…”

“…CSFV infection inhibits dsRNA-induced apoptosis at a number of apoptotic checkpoints, preventing caspase-8 activation and loss of mitochondrial membrane potential (Johns et al, 2010). N pro contributes to the antagonism of dsRNA-mediated apoptosis; however the underlying mechanism has not been determined (Johns et al, 2010;Ruggli et al, 2003Ruggli et al, , 2005. In this study, no decrease in HAX-1 levels was observed in PK-15 cells following treatment with dsRNA, indicating that N pro does not inhibit dsRNA-mediated apoptosis by preventing HAX-1 degradation.…”

“…N pro antagonizes dsRNA-mediated apoptosis (Johns et al, 2010;Ruggli et al, 2003Ruggli et al, , 2005. To determine whether HAX-1 is also degraded during dsRNA-induced apoptosis, we treated parental PK-15 cells and a PK-15 cell line stably expressing N pro with dsRNA (100 mg pIpC ml 21 added directly to the medium), and then analysed whole-cell extracts by Western blot.…”

“…N pro contributes to this inhibition by targeting IFN regulatory factor-3 for proteasomal degradation (Bauhofer et al, 2007;Chen et al, 2007;Hilton et al, 2006;Seago et al, 2007). N pro also plays a role in the evasion of dsRNA-induced apoptosis (Johns et al, 2010;Ruggli et al, 2003Ruggli et al, , 2005.…”

The Ribonucleotide Reductase R1 Subunits of Herpes Simplex Virus 1 and 2 Protect Cells against Poly(I · C)-Induced Apoptosis

“…This study reveals that, in addition, HSV R1s protect HSV-infected cells against poly(I ⅐ C)-induced caspase 8 activation, possibly through their interaction with RIP1 and caspase 8. Interestingly, this type of viral protection is not restricted to HSV, since a recent study showed that infection with the classical swine fever virus, a positive-sense RNA virus, protects endothelial cells from extracellular poly(I ⅐ C)-mediated apoptosis, notably by inhibiting caspase 8 activation (34). Thus, viral inhibition of dsRNAmediated apoptosis could be a strategy used by several viruses to ensure host cell survival and, therefore, efficient replication and spreading.…”