key: cord-0259180-v8ri13vz authors: Gao, Bo; Gong, Xiaoqian; Fang, Shouguo; Weng, Wenlian; Sun, Yingjie; Meng, Chunchun; Tan, Lei; Song, Cuiping; Qiu, Xusheng; Liu, Weiwei; Forlenza, Maria; Ding, Chan; Liao, Ying title: Inhibition of Anti-viral Stress Granule Formation by infectious bronchitis virus endoribonuclease nsp15 Ensures Efficient Virus Replication date: 2020-06-11 journal: bioRxiv DOI: 10.1101/2020.06.11.145862 sha: 1d6c3e87289dd9392b12fa5218b726c2b3b6712f doc_id: 259180 cord_uid: v8ri13vz 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 virus, coronavirus, mutates frequently and circulates among animals, imposing great threat to public safety and animal health; however, the significance of SGs in coronavirus infections 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 provide 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 show that the intrinsic endoribonuclease activity of nsp15 is responsible for the inhibition 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 triggered transcription of IFN-β which in turn greatly affected recombinant virus 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 PRRs and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV suppresses 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 speculate that coronaviruses employ similar mechanisms to antagonize the host anti-viral SGs formation for efficient virus replication, as the endoribonuclease function of nsp15 is conserved in all coronaviruses. Author summary It has been reported that stress granules (SGs) are part of the host cell antiviral response. Not surprisingly, viruses in turn produce an array of antagonists to counteract such host response. Here, we show that IBV inhibits the formation of SGs through its endoribonuclease nsp15, by reducing the accumulation of viral dsRNA, evading the activation of PKR, and by subsequently inhibiting eIF2α phosphorylation and SGs formation. Nsp15 also inhibits SG formation independent of the eIF2α pathway, probably by targeting host mRNA. Depletion of SG scaffold proteins decreases IRF3-IFN response and increases the production of IBV. All coronaviruses encode a conserved endoribonuclease nsp15, and it will be important to determine whether also other (non-avian) coronaviruses limit the formation of anti-viral SGs in a similar manner. In this study, we employed chicken fibroblast DF-1 cells in most experiments; in 139 specific cases, to facilitate the detection of cellular proteins, and due to the 140 unavailability of antibodies against chicken proteins of interest (see Table 1 ), we also 141 included mammalian H1299 and Vero cells lines, which also support replication of the SGs positive cells, another SGs marker, TIAR, was found to colocalize with G3BP1 155 granules, altogether demonstrating that IBV induces canonical SGs (Fig 1D-E formation not only in chicken cells but also in two different mammalian cell lines, due 169 to the availability of antibodies directed against mammalian proteins and their lack of 170 cross-reactivity to the chicken proteins of interest, we next proceeded with H1299 and 171 Vero cells. In IBV-infected H1299 cells, PKR and eIF2α phosphorylation was 172 comparable to that observed in mock infected cells (Fig 2A) . In Vero cells, however, 173 PKR, but not eIF2α, phosphorylation was slightly increased at 20 and 24 h.p.i. (Fig 2B) , altogether suggesting that the PKR-eIF2α-SGs pathway is not obviously triggered by 175 IBV infection. In parallel, we did not observe any cleavage product of either G3BP1 or 176 TIA-1 throughout IBV infection in both H1299 and Vero cells (Fig 2) , altogether 177 indicating that IBV may avoid PKR activation to prevent SGs formation. reasons (data not shown). We next explored whether IBV interfered with the 196 phosphorylation of eIF2α triggered by sodium arsenite or heat shock. We observed a 197 significant upregulation of phospho-eIF2α by sodium arsenite or heat shock treatment 198 in H1299 cells; however, there was no reduction of phospho-eIF2α by IBV infection 199 (Fig 3D-E) . Collectively, these data indicate that IBV infection restricts both eIF2α-200 dependent and -independent SG formation, probably by interfering with SG assembly 201 or disassembly and not with direct eIF2α phosphorylation. Fig. 4A . In cells expressing nsp2, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, 209 nsp12, nsp16, 3b, E, 5a, 5b, M, or N, SGs formation remained intact (Fig 4B) , 210 suggesting that alone these proteins have no inhibitory effect on the formation of SGs. Interestingly, only in nsp15-expressing cells, the heat shock-induced SGs were absent 212 (as indicated by white arrow), suggesting that nsp15 may be the viral protein 213 responsible for efficient suppression of SGs formation. We also investigated, but failed 214 to detect, the expression of nsp3, nsp10, nsp13, nsp14, S, and 3a, therefore it cannot be 215 excluded that also these viral proteins might be involved in inhibition of SG formation. These results however demonstrate that nsp15 alone is sufficient to block SGs 217 formation. Nsp15 is a conserved endoribonuclease of coronaviruses. It has been reported that rIBV-nsp15-H238A in which the nsp15 catalytic site H238 was replaced with an 244 alanine ( Fig 6A) . We also constructed, but failed to recover nsp15-defective rIBV- [81]. In the current study, when nsp15 was overexpressed, it was worth noting that 451 nsp15 located to nucleus (Fig 4, Fig 5A-C) . Taken all our observations together, we 452 speculate that nuclear nsp15 may interfere with the host pre-mRNA processing or The plasmids encoding IBV nsp2, nsp4, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp15, 514 nsp16, 3b, E, 5a, 5b, M and N were generated by amplification of cDNA from IBV 515 Beaudette-infected Vero cells using corresponding primers ( Table 1) After 48 h.p.i., puromycin (2 μg/ml) was applied to select for G3BP knockout cells. 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