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  • br Materials and Methods br Results


    Materials and Methods
    Discussion Viral infection of eukaryotic HG-10-102-01 activate signaling pathways both via specific interaction with pattern recognition receptors (TLRs, RIG-I, MDA5) and more nonspecific mechanisms such as accumulation of newly synthesized viral glycoproteins in the ER, leading to UPR. These pathways may induce interferon production, protein synthesis attenuation, cellular apoptosis or a range of other responses to curb viral production (Langevin et al., 2013). ISAV is a salmon pathogen causing serious disease problems in aquaculture of this species, both in Europe and the Americas. Previous reports have demonstrated that this virus may induce either apoptosis (Joseph et al., 2004, Schiotz et al., 2009) or autophagy (Schiotz et al., 2010), dependent both on cell type and viral strain. However, since the mechanisms behind these responses to ISAV infection is not clear, we investigated the possibility that ISAV induce UPR, a known inducer for both apoptosis and autophagy during viral infection (Lee et al., 2009, Li et al., 2013). Examination of the ultrastructure in ISAV infected cells revealed dilated ER similar to cisternae often observed during virally induced UPR (e.g., HCV infections) (Asselah et al., 2010). Previous reports have shown that ER volume may increase up to fivefold during UPR and is driven by lipid biosynthesis. The increased ER volume is in itself an important part of the UPR response, making protein aggregation (due to misfolding) less likely and to accommodate the increased synthesis of ER chaperones (Schuck et al., 2009). However, at the light microscopic level, the intracellular redistribution of the ATP-sensitive K+ channels visualized by fluorescent Glibenclamide staining of thapsigargin treated cells was not reproduced in infected cells. This may be due to the more subtle induction of UPR by viral infection or for kinetic reasons. ER stress induced by viral infection may activate UPR via one, two or all three main signaling pathways: IRE1, ATF6 and PERK. UPR signaling is also dependent on the infection stage as exemplified by dengue virus, that initially activate PERK but at later stages supresses this pathway (Pena and Harris, 2011). In line with the ultrastructural changes seen in ISAV infected cells in vitro we could demonstrate that the IRE1 pathway was activated, as a transient XBP1 splicing activity could be demonstrated. Viral induction of this pathway was delayed compared to drug treatment and this in in line with results obtained with other UPR inducing viruses (Li et al., 2013). Although not completely overlapping, the combined results from transcriptional analyses of infected cells in vitro and in vivo, suggests that all 3 main signaling pathways for UPR were activated by ISAV. Upregulation of XBP1is mainly controlled by XBP1 itself and ATF6 (Yoshida et al., 2001), XPB1 splicing by IRE1 and finally, GADD34 upregulation is mainly (but not solely) controlled via the PERK pathway and constitutes a negative feedback loop via its activity as a regulatory subunit of PP1 (that dephosphorylates eIF2a) (Novoa et al., 2001). This negative feedback loop may therefore partly explain why hyperphosphorylation of eIF2a was weak and translation seemed relatively unperturbed in infected cells (where GADD34 was steadily increasing), compared to drug treated cells (displaying a short burst of GADD34 mRNA induction returning to normal within 24 h). Several other viruses have been shown to inhibit the eIF2a pathway to ensure translation of viral proteins during infection (Jheng et al., 2014, Li et al., 2013). The importance of GADD34 for innate immunity against viral infection was recently demonstrated by the increased susceptibility of GADD34 −/− fibroblasts and neonate mice to Chikungunya virus infections (Clavarino et al., 2012). ISAV infection therefore seem to induce sufficient eIF2a phosphorylation for activation of ATF4 translation to occur (and subsequent GADD34 transcription), but without attenuating production of interferon and cellular ISGs (Robertsen, 2006, Robertsen, 2008). The activation of IRE1 pathway also displayed a delayed kinetics for ISAV compared to induction by pharmacological treatment. The most striking difference between classical UPR as induced by pharmacological agents such as thapsigargin and tunicamycin was the lack of effect of ISAV on expression of GRP78, a classical marker of UPR (Ron and Walter, 2007). However, this is in agreement with the latest views on the distinction between a classical UPR (due to accumulation of unfolded proteins in ER) and what is now called the MSR (for Microbial Stress Response) (Claudio et al., 2013). This response may attenuate translation but without completely blocking synthesis of cytokines and antiviral proteins (ISGs), also in line with previous studies in ISAV demonstrating a strong positive effect on ISG transcription (Schiøtz et al., 2009).