Coronavirus infection induces the unfolded protein response (UPR), a cellular signalling pathway composed of three branches, triggered by unfolded proteins in the endoplasmic reticulum (ER) due to high ER load. We have used RNA sequencing and ribosome profiling to investigate holistically the transcriptional and translational response to cellular infection by murine hepatitis virus (MHV), often used as a model for the Betacoronavirus genus to which the recently emerged SARS-CoV-2 also belongs. We found the UPR to be amongst the most significantly up-regulated pathways in response to MHV infection. To confirm and extend these observations, we show experimentally the induction of all three branches of the UPR in both MHV- and SARS-CoV-2-infected cells. Over-expression of the SARS-CoV-2 ORF8 or S proteins alone is itself sufficient to induce the UPR. Remarkably, pharmacological inhibition of the UPR greatly reduced the replication of both MHV and SARS-CoV-2, revealing the importance of this pathway for successful coronavirus replication. This was particularly striking when both IRE1α and ATF6 branches of the UPR were inhibited, reducing SARS-CoV-2 virion release (~1,000-fold). Together, these data highlight the UPR as a promising antiviral target to combat coronavirus infection.

In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro. Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway.IMPORTANCE Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6Pro. This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-b-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake. INTRODUCTIONThe family Caliciviridae is divided into four genera: Vesivirus, Lagovirus, Sapovirus and Norovirus. The human noroviruses are the most common cause of acute viral gastroenteritis, especially in industrialized countries (Lopman et al., 2003); there is currently no treatment or vaccine for these viruses. In addition, there is still no routine tissue culture system for the propagation of human noroviruses, but the recent discovery of murine norovirus-1 (MNV-1) has provided an efficient model system for the study of norovirus replication, as this virus replicates efficiently in murine macrophages and dendritic cells (DCs) (Karst et al., 2003;Wobus et al., 2004). MNV-1 is highly prevalent in laboratory mice and has been shown to be lethal to mice with impaired innate immunity (Hsu et al., 2006;Karst et al., 2003;Muller et al., 2007).Noroviruses are non-enveloped and contain a singlestranded RNA genome of about 7.3 kb, which is linked to the viral VPg protein at the 59 end and is polyadenylated at the 39 end (Karst et al., 2003;Wobus et al., 2004). The genome is organized into four open reading frames (ORF-1-4). ORF-1 encodes the non-structural proteins, whereas ORF-2 and ORF-3 encode structural proteins (Sosnovtsev et al., 2006). ORF-4 was recently identified within the MNV-1 genome and encodes a single protein of unknown function (Thackray et al., 2007).For many viruses, infection requires virus uptake by endocytosis. A number of different endo...

RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3′ UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as anti-viral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The anti-viral potential of this conserved element represents a promising start for further research into targeting conserved elements in RNA viruses.

Porcine Epidemic Diarrhea Virus (PEDV) is a member of the genus Alphacoronavirus, in the family Coronaviridae, of the Nidovirales order and outbreaks of porcine epidemic diarrhoea (PED) were first recorded in England in the 1970s. Intriguingly the virus has since successfully made its way around the globe, while seemingly becoming extinct in parts of Europe before its recent return from Northern America. In this review we are re-evaluating the spread of PEDV, its biology and are looking at lessons learnt from both failure and success. While a new analysis of PEDV genomes demonstrates a wider heterogeneity of PEDV than previously anticipated with at least five rather than two genotypes, biological features of the virus and its replication also point towards credible control strategies to limit the impact of this re-emerging virus.