Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

Xufang Deng; Yafang Chen; Anna M. Mielech; Matthew Hackbart; Kristina R. Kesely; Robert C. Mettelman; Amornrat O’Brien; Mackenzie E. Chapman; Andrew D. Mesecar; Susan C. Baker

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/782409 doi: bioRxiv preprint designated the virus as DUBmut. Upon evaluating virus replication of the DUBmut virus by 175 performing a growth kinetics experiment in parallel with wild-type virus, we found that the 176 DUBmut virus replicates with essentially identical kinetics as the wild-type in a murine 177 astrocytoma cell line (DBT cells) (Fig. 4A ). These results are consistent with previous studies of 178 coronavirus interferon antagonists, which showed in many cell lines that viral-mediated interferon 179 antagonism is not essential for virus replication (5, 6). Regarding the other ubiquitin-interacting 180 residues identified in the structural analysis, we attempted to rescue virus with substitutions at the 181 F1812 position, but were unable to recover viable virus. These results indicate that F1812 may 182 play a critical role within the polyprotein during virus replication. We were able to recover virus 183 containing the R1803A substitution, but found that it had no detectable phenotype, which we 184 documented in our previous study (5). Here, we focus our efforts on evaluating replication and IFNï¡ï€ protein into the supernatant, as detected by ELISA (Fig. 4B ). We show that this activation 191 of IFN is dependent on expression of pattern recognition receptor MDA5 (Fig. 4D ), in agreement 192 with previous reports (5, 6, 33). To our surprise, we found that replication of DUBmut is not 193 impaired relative to the wild-type virus in BMDMs, as measured by level of nucleocapsid RNA 194 (Fig. 4E ) and evaluation of infectious virus particle production over time in the kinetics assay (Fig. 195 4F). These results demonstrate that an elevated interferon response is generated during replication 196 All rights reserved. No reuse allowed without permission.