Probing the unfolded protein response to mouse hepatitis coronavirus infection through RNA sequencing and ribosome profiling

Georgia M. Cook; Katherine Brown; Krzysztof Franaszek; Nathan A. Moore; Stuart G. Siddell; Ian Brierley; Andrew E. Firth; Nerea Irigoyen

Xbp-1u and Xbp-1s in virus-infected cells, we inspected the ribosome profiling data (Fig 3B) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/292979 doi: bioRxiv preprint GSK-2606414 (PERKi) is a potent and selective high affinity ligand of the PERK kinase, that 310 interferes with kinase activity by competing for ATP [56, 57] . In MHV-infected 17 Cl-1 cells at 5 311 and 8 h p.i., the drug prevented autophosphorylation of PERK (Fig 6A, lower panel) and 312 phosphorylation of the PERK substrate, eIF2α, in a dose-dependent manner (Fig 6A, upper panel) , 313 effectively blocking this branch of the UPR. Pulse labelling of infected cells for one hour at 5 h p.i. 314 revealed, as expected, that prevention of eIF2α phosphorylation increased modestly both viral (Fig 315 6A ) and host protein synthesis (Fig 6B) , without effect on mock-infected cells (Fig 6B) . Rpl19 was only slightly increased at the highest PERKi concentrations (Fig 6F, upper panel) . (with fewer than ten counts from all samples combined) were discarded, following which read 498 counts were normalised by the total number of reads mapping to host mRNA for that library. This 499 . CC-BY-NC-ND 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/292979 doi: bioRxiv preprint 1 means the very large amount of vRNA present in infected samples should not affect the analyses. 500