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  • In conclusion we provide convincing evidence


    In conclusion, we provide convincing evidence that the PRRSV-induced SGs are indeed bona fide SGs. While we determined that mRNA is present in the PRRSV-induced SGs, we did not distinguish its origin. Future studies will need to determine whether the mRNA glucagon receptor stored in PRRSV-induced SGs is of cellular or viral origin. It is interesting to speculate that specific RNA transcripts are recruited to the PRRSV-induced SGs in order to regulate translation of either host or viral mRNAs. We also identified the SG components G3BP1, G3BP2 and USP10 are dispensable for PRRSV replication. Ultimately, our data suggests that the PRRSV-induced SGs may function to suppress host cellular translation, however, further experiments are needed to definitively support this hypothesis. Indeed, monitoring puromycylation under G3BP1/2 knock-down condition in the context of PRRSV infection would inform whether the PRRSV-induced SGs regulate host cellular translation. Furthermore, the fate of RNA transcripts sequestered in the PRRSV-induced SGs remains to be determined. There are many important but unanswered questions that are beyond the scope of this initial study. For example, are the transcripts being stored temporarily in SGs, or transferred to P-bodies for degradation? Are specific transcripts recruited to SGs during PRRSV infection? A better understanding of the molecular events involved in the formation of PRRSV-induced SGs will undoubtedly help understand the molecular mechanism of PRRSV pathogenesis.
    Conflict of interest
    Acknowledgements This work was supported by a pre-doctorial fellowship grant to Nicholas Catanzaro from the U.S. Department of Agriculture National Institute of Food and Agriculture (USDA NIFA 2017-67011-26045). The authors thank the expert assistance of C. Lynn Heffron, Shannon Matzinger, Sakthivel Subramaniam, Debin Tian, and Qian Cao for their help and guidance with this work.
    Introduction Infectious salmon anemia virus is an aquatic orthomyxovirus of the genus Isavirus, causing high mortalities and great economic losses in the Atlantic salmon (Salmo salar L.) farming industry (Christie et al., 1991, Dannevig and Falk, 1994, Falk et al., 1997, Mjaaland et al., 1997). The virus is reported to cause acute or protracted disease in fish (Mjaaland et al., 2005). Several strains of ISAV are known and many differ in the highly polymorphic region of the hemagglutinin-esterase protein. This region also affects the ability of the virus to induce acute versus protracted disease in affected fish (Devold et al., 2001, Mjaaland et al., 2005). Although much is known about the structure and genetics of the virus, less is known about the route of entry, pathogenesis and immune reactions induced by ISAV. Recent studies suggest that the virus may enter the host through the gill epithelia (Weli et al., 2013) and via blood spread to endothelial cells (Aamelfot et al., 2012) where replication has been observed. ISAV binds to mucin type glucagon receptor (Eliassen et al., 2000b, Falk et al., 1997) such as 4-0-acetylated sialic acids (Hellebo et al., 2004). Inside cells, ISAV elicits various forms of biochemical (Schiøtz et al., 2009, Schiøtz et al., 2010) and transcriptional stress responses (Jorgensen et al., 2008, Lauscher et al., 2011, LeBlanc et al., 2010, Li et al., 2011, Schiotz et al., 2008), but there are to date no reports suggesting involvement of the unfolded protein stress response (UPR) during ISAV infection. When animals are infected with viruses a wide range of innate and adaptive immune responses are activated. Viral infection elicits broad changes in the type and amounts of protein made by the infected cell and thereby disrupt cellular homeostasis. In addition to production of viral proteins cells will begin to make antiviral proteins and release cytokines such as type I interferons that will bind to receptors on infected and noninfected neighboring cells and activate transcription of interferon-stimulated genes (ISGs) (Versteeg and Garcia-Sastre, 2010). These messengers will make them more resistant to infection and thereby restrict the spread of virus. Inside the already infected cells, synthesis of viral protein takes place in parallel with induction of antiviral proteins. These responses will either clear the virus or the cells will enter an apoptotic programme leading to its death by apoptosis (to the benefit of the organism). One important cellular homeostatic process known to be activated during various forms of viral infections is the unfolded protein response (UPR), or ER-stress response. UPR is found in all eukaryotic organisms and is induced upon accumulation of unfolded proteins in the ER due to overload (e.g., under viral infections) but also other forms of cellular stress e.g., perturbation of calcium homeostasis and chemical oxidants (Hetz, 2012). Three resident ER membrane proteins are the main regulators of UPR: activating transcription factor 6 (ATF6), inositol requiring enzyme 1 (IRE1 a ribonuclease/kinase) and PKR such as ER kinase (PERK) (Supplementary figure 1). These three ER membrane proteins are normally held inactive by interaction of their luminal tails with the ER chaperone protein BiP. Upon accumulation of unfolded proteins in the ER lumen, BiP is released from the UPR regulators (ATF6, IRE1 and PERK) leading to their activation (Hetz, 2012). It was demonstrated more than 10 years ago that viral infection can induce UPR (Waris et al., 2002) and later this has been confirmed for a range of viruses (Ambrose and Mackenzie, 2011, Barry et al., 2010, Huang et al., 2011, Pasqual et al., 2011, Zhang et al., 2010). The replication of viruses may either be inhibited or facilitated upon induction of UPR (Zhang and Wang, 2012). Influenza A virus, (another orthomyxovius) has also recently been demonstrated to modulate the UPR response in infected cells (Hassan et al., 2012). Accumulating evidence indicated that ER stress modulates immune responses (such as PERK mediated inhibition of type I IFN signaling) and therefore also viral pathogenesis (Minakshi et al., 2009). Since nothing is known about the role of UPR in ISAV replication in salmon cells in vitro or in vivo we here report the potential role of these pathways during ISAV infections.