We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (N pro ) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from N pro -expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.

Macroautophagy/autophagy delivers damaged proteins and organelles to lysosomes for degradation, and plays important roles in maintaining tissue homeostasis by reducing tissue damage. The translocation of LC3 to the limiting membrane of the phagophore, the precursor to the autophagosome, during autophagy provides a binding site for autophagy cargoes, and facilitates fusion with lysosomes. An autophagy-related pathway called LC3-associated phagocytosis (LAP) targets LC3 to phagosome and endosome membranes during uptake of bacterial and fungal pathogens, and targets LC3 to swollen endosomes containing particulate material or apoptotic cells. We have investigated the roles played by autophagy and LAP in vivo by exploiting the observation that the WD domain of ATG16L1 is required for LAP, but not autophagy. Mice lacking the linker and WD domains, activate autophagy, but are deficient in LAP. The LAP −/mice survive postnatal starvation, grow at the same rate as littermate controls, and are fertile. The liver, kidney, brain and muscle of these mice maintain levels of autophagy cargoes such as LC3 and SQSTM1/p62 similar to littermate controls, and prevent accumulation of SQSTM1 inclusions and tissue damage associated with loss of autophagy. The results suggest that autophagy maintains tissue homeostasis in mice independently of LC3-associated phagocytosis. Further deletion of glutamate E230 in the coiled-coil domain required for WIPI2 binding produced mice with defective autophagy that survived neonatal starvation. Analysis of brain lysates suggested that interactions between WIPI2 and ATG16L1 were less critical for autophagy in the brain, which may allow a low level of autophagy to overcome neonatal lethality.

Infection with virulent strains of classical swine fever virus (CSFV) results in an acute haemorrhagic disease of pigs, characterized by disseminated intravascular coagulation, thrombocytopenia and immunosuppression, whereas for less virulent isolates infection can become chronic. In view of the haemorrhagic pathology of the disease, the effects of the virus on vascular endothelial cells was studied by using relative quantitative PCR and ELISA. Following infection, there was an initial and short-lived increase in the transcript levels of the proinflammatory cytokines interleukins 1, 6 and 8 at 3 h followed by a second more sustained increase 24 h post-infection. Transcription levels for the coagulation factor, tissue factor and vascular endothelial cell growth factor involved in endothelial cell permeability were also increased. Increases in these factors correlated with activation of the transcription factor NF-kB. Interestingly, the virus produced a chronic infection of endothelial cells and infected cells were unable to produce type I interferon. Infected cells were also protected from apoptosis induced by synthetic ouble-stranded RNA. These results demonstrate that, in common with the related pestivirus bovine viral diarrhoea virus, CSFV can actively block anti-viral and apoptotic responses and this may contribute to virus persistence. They also point to a central role for infection of vascular endothelial cells during the pathogenesis of the disease, where a proinflammatory and procoagulant endothelium induced by the virus may disrupt the haemostatic balance and lead to the coagulation and thrombosis seen in acute disease.