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

The Coronaviridae are a family of enveloped viruses with positive-sense, monopartite, single-46 stranded RNA genomes. At 27-32 kb, coronaviruses (CoVs) have the largest known RNA 47 lysis, caused stress-induced accumulation of ribosomes at the 5´ end of coding regions (CDSs; 103 [16] ). In this work, therefore, cells were not pretreated with CHX and were snap-frozen before lysis, 104 which avoids this artefact ( Table 1 ). Some of the most differentially transcribed cellular genes (ochre 113 points) are related to the host translational apparatus: Rplp1 -a ribosomal protein from the large 114 subunit; Eef1a1 -eukaryotic elongation factor 1A-1; Rps21 -a ribosomal protein from the small 115 subunit; Eif3f -eukaryotic initiation factor 3 subunit F; Eif3j1 -eukaryotic initiation factor 3 116 subunit J; and Eif2b3. This is also reflected in the gene ontology (GO) term enrichment analysis 117 ( Fig 1B; full results in Supplementary Table 2), which reveals that all GO terms enriched in the list 118 of genes significantly transcriptionally down-regulated in infection were related to protein synthesis 119 (blue points). 120 121 Several transcription-related genes were found to be transcriptionally up-regulated, for example 122 Polr2a, the gene coding for the largest subunit of RNA polymerase II (Fig 1A) . GO terms related to 123 transcription, for example "transcription by RNAPII" (GO:0006366), are also enriched in the up-124 regulated genes list. Many histones feature in the transcriptionally up-regulated gene list and, as 125 such, many histone-related GO terms are enriched in this list (Fig. 1B) . Significantly, the GO term 126 "response to unfolded protein" (GO:0006986) is enriched 4.85-fold in the list of genes 127 transcriptionally up-regulated during infection (p=0.046, FDR-adjusted p-value), with similar fold 128 6 changes observed for "response to topologically incorrect protein" (GO:0035966; 4.31-fold 129 enrichment; p=0.046) (Supplementary Table 2 ) and "response to endoplasmic reticulum stress" 130 (GO:0034976; 3.8-fold enrichment; p=0.012) (Fig 1B - note that these terms are clustered within 131 the "response to unfolded protein" GO term). Accordingly, some of the most differentially 132 expressed genes were involved in the UPR such as Herpud1 -homocysteine inducible ER protein 133 with ubiquitin like domain 1; Bip -immunoglobulin heavy chain-binding protein; Chac1 -134 glutathione-specific gamma-glutamylcyclotransferase 1; Chop -a C/EBP family transcription factor 135 involved in the ER stress response, and Xbp1 -X-Box Binding Protein 1 ( Fig 1A) . L19 (Rpl19), which has been reported to be unaffected by ER stress [22, 23] . Up-regulated 145 transcripts had qRT-PCR values broadly consistent with the RNASeq measurements ( Fig 1A and 146 1C) whereas there was a little more variation in the down-regulated transcripts, which may be partly 147 explained by the observation that Rpl19 itself was slightly, though not statistically significantly, 148 down-regulated (log 2 (fold change) = -0.34, p=0.37) (Fig 1A, yellow) . 149 150