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 an 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, and 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.
Coronavirus encodes the conserved endoribonuclease nsp15, which has been reported to antagonize IFN responses by mediating evasion of recognition by dsRNA sensors. SGs are part of the host cell anti-viral response; not surprisingly, viruses in turn produce an array of antagonists to counteract such host response. Here, we show that IBV prevents the formation of SGs via nsp15, by reducing the accumulation of viral dsRNA, thereby evading the activation of PKR, phosphorylation of eIF2α, and formation of SGs. Depletion of SG scaffold proteins G3BP1/2 decreases IRF3-IFN response and increases the production of IBV. When overexpressed alone, nsp15s from different coronaviruses (IBV, PEDV, TGEV, SARS-CoV, and SARS-CoV-2) interferes with chemically- and physically-induced SGs, probably by targeting essential SGs assembly factors. In this way, coronaviruses antagonize the formation of SGs by nsp15, via reducing the viral dsRNA accumulation and sequestering/depleting critical component of SGs. To our knowledge, this is the first report describing the role of coronavirus nsp15 in the suppression of integral stress response, in crosstalk with anti-innate immune response.