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.
The G3BP1 and G3BP2 stably knockout cells were obtained after 5-6 passages and the 608 absence of G3BP1/2 expression was confirmed by Western blot analysis and genome 609 sequencing. Table 2 ). The Bsa I/BsmB I digested products 