1 1 Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus 2 suppresses type I and type III interferon responses 3 4 Xufang Denga, Albert van Geelenb, Alexandra C. Buckleyb, Amornrat OBriena, Angela 5 Pillatzkic, Kelly M. Lagerb, Kay S. Faabergb*, and Susan C. Bakera* 6 7 aDepartment of Microbiology and Immunology, Loyola University Chicago, Stritch 8 School of Medicine, Maywood, IL 60153, USA; 9 bVirus and Prion Research Unit, USDA-ARS-National Animal Disease Center, Ames, IA 10 50010, USA. 11 cAnimal Disease Research however, individual variation was 237 observed in all groups. Representative images of ileum sections from an age-matched, 238 uninfected piglet and from one piglet per virus-infected group are shown in Figure 7, 239 on February 6, 2019 by guesthttp:/jvi.asm.org/Downloaded from 12 upper panel. To determine if the sites of virus replication were similar in all infected 240 animals, we performed immunohistochemistry to detect PEDV nucleocapsid protein. 241 Similar to previous studies (2426), viral antigen was detected in epithelial cells, and we 242 found that the sites of replication of the engineered viruses were essentially identical to 243 those found with PEDV-Colorado (Figure 7, lower panel). 244 245 Discussion 246 Our study reveals that coronaviruses encode a highly conserved enzyme, EndoU, 247 that mitigates type I and type III IFN responses in virus-infected macrophages and 248 epithelial cells. By constructing an infectious clone of the virulent Colorado strain of 249 PEDV, and an isogenic strain with a mutation that inactivated EndoU activity, we could 250 directly compare the replication kinetics and innate immune responses of these two 251 viruses in cell culture and in animals. Consistent with previous studies of murine 252 coronavirus (12, 13), we found that both wild-type and the EndoU-mutant PEDV 253 replicate efficiently in interferon deficient Vero cells. However, the loss of EndoU activity 254 correlated with activation of type I (IFN-/) and type III (IFN-) IFN responses in 255 macrophages and epithelial cells, and reduced viral titer in PK-1 cells. These results are 256 also in line with previous reports indicating that replication of SARS-CoV is enhanced in 257 mice lacking the IFN- receptor (34, 35), and that PEDV replication can be inhibited by 258 treatment of IFN-in epithelial cells (36, 37). Overall, these studies illustrate the critical 259 role of IFNs in controlling enteric pathogens, and that coronaviruses have evolved 260 mechanisms to evade these host innate immune defenses. 261 on February 6, 2019 by guesthttp:/jvi.asm.org/Downloaded from 13 Our study illustrates that the innate immune response to PEDV is cell type 262 specific, with macrophages responding via IFN-/ response, whereas the epithelial cell 263 response includes IFN-. It is important to note that infection with wild-type PEDV does 264 elicit IFN responses from epithelial cells and macrophages, but the timing of the IFN 265 response is delayed and the magnitude of IFN levels is lower compared to PEDV-266 EndoU mutant virus infection (Fig. 3). The earlier IFN response elicited by icPEDV-267 EnUmt infection results in the transcription of a series of ISGs that are known to induce 268 an antiviral state and thereby limit propagation of the invading pathogen. We also report 269 detection of reduced virus shedding and mortality in piglets infected with icPEDV-EnUmt 270 compared to piglets infected with the isogenic wild-type strain (Fig. 6B and C). These 271 results suggest that activation of an early innate immune response alters virus 272 replication and pathogenesis, but additional in vivo research is needed to fully 273 investigate this issue. For example, the in vivo response to CoV infection may differ, 274 depending on the dose of the virus and the age of the animal. It will also be important to 275 evaluate the frequency of reversion of EndoU mutants and to employ strategies to make 276 these viruses recombination-resistant (38). It is possible that the pathology we detected 277 at day 7 in the icPEDV-EnUmt-infected animals could be generated by revertant viruses 278 (Fig. 7). Future studies using viruses containing multiple mutations in EndoU, thus less 279 likely to revert, should be evaluated at multiple time points to determine the extent of 280 virus replication and the local immune response. For coronaviruses that encode multiple 281 IFN antagonists, it will be interesting to evaluate the role of each antagonist on the type 282 I and type III IFN responses, and to determine if inactivating a constellation of 283 antagonists, perhaps both the highly conserved nonstructural protein antagonists and 284 on February 6, 2019 by guesthttp:/jvi.asm.org/Downloaded from 14 accessory proteins, may be necessary to fully attenuate endemic and emerging strains 285 (29, 39, 40). 286 Evaluating type III IFN