Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-β which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.
Protein kinase R (PKR) is a critical host restriction factor against invading viral pathogens. However, this molecule is inactivated in the cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), an economically devastating pathogen to the world swine industry. Here, we report that this event is to suppress cellular inflammation and is mediated by the viral replicase protein nsp1β. We show that nsp1β is a stress-responsive protein, enters virus-induced stress granules (SGs) during infection, and repurposes SGs into a proviral platform, where it co-opts the SG core component G3BP1 to interact with PKR in a regulated manner. RNA interference silencing of G3BP1 or mutation of specific nsp1β residues (VS19GG) can abolish the antagonization of PKR activation. The viral mutant carrying the corresponding mutations induces elevated level of PKR phosphorylation and pronounced production of inflammatory cytokines (e.g., tumor necrosis factor-α, interleukin [IL]-6, and IL-8), whereas small-interfering RNA knockdown of PKR or treatment with C16, a PKR inhibitor, blocks this effect. Thus, PRRSV has evolved a unique strategy to evade PKR restriction to suppress host inflammatory responses.
2Cytoplasmic stress granules (SGs) are generally triggered by stress-induced 3 translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-4 viral functions due to their involvement in protein synthesis shut off and recruitment of 5 innate immune signaling intermediates. The largest RNA virus, coronavirus, mutates 6 frequently and circulates among animals, imposing great threat to public safety and 7 animal health; however, the significance of SGs in coronavirus infections is largely 8 unknown. Infectious bronchitis virus (IBV) is the first identified coronavirus in 1930s 9 and has been prevalent in poultry farm for many years. In this study, we provide 10 evidence that IBV overcomes the host antiviral response by inhibiting SGs formation 11 via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we 12 observed that IBV infection not only did not trigger SGs formation in approximately 13 80% of the infected cells, but also impaired the formation of SGs triggered by heat 14 shock, sodium arsenite, or NaCl stimuli. We show that the intrinsic endoribonuclease 15 activity of nsp15 is responsible for the inhibition of SGs formation. In fact, nsp15-16 defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, 17 along with accumulation of dsRNA and activation of PKR, whereas wild type IBV 18 failed to do so. Consequently, infection with rIBV-nsp15-H238A triggered 19 transcription of IFN-β which in turn greatly affected recombinant virus replication. 20Further analysis showed that SGs function as antiviral hub, as demonstrated by the 21 attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. 22Additional evidence includes the aggregation of PRRs and signaling intermediates to 23 the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease 24 nsp15 of IBV suppresses the formation of antiviral hub SGs by regulating the 25 accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually 26 ensuring productive virus replication. We speculate that coronaviruses employ similar 27 mechanisms to antagonize the host anti-viral SGs formation for efficient virus 28 replication, as the endoribonuclease function of nsp15 is conserved in all coronaviruses. 29 3 30Author summary 31 It has been reported that stress granules (SGs) are part of the host cell antiviral 32 response. Not surprisingly, viruses in turn produce an array of antagonists to counteract 33 such host response. Here, we show that IBV inhibits the formation of SGs through its 34 endoribonuclease nsp15, by reducing the accumulation of viral dsRNA, evading the 35 activation of PKR, and by subsequently inhibiting eIF2α phosphorylation and SGs 36 formation. Nsp15 also inhibits SG formation independent of the eIF2α pathway, 37 probably by targeting host mRNA. Depletion of SG scaffold proteins decreases IRF3-38 IFN response and increases the production of IBV. All coronaviruses encode a 39 conserved endoribonuclease nsp15, and it will be i...
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