Fig 1.
Ribosome profiling reveals the unfolded protein response as a key pathway in the host response to MHV-A59 infection.
(A) Schematic of the three branches of the UPR (IRE1α, ATF6, and PERK). ERAD = ER-associated protein degradation. (B) Top five most significantly enriched Reactome pathways [11] associated with the lists of transcriptionally up-regulated genes (orange triangles pointing upwards) and transcriptionally down-regulated genes (blue triangles pointing downwards), plotted according to the false discovery rate (FDR)-corrected p value of the enrichment. Full results, including pathway IDs, are in S3 Table. (C) Volcano plot showing the relative change in abundance of cellular transcripts and the FDR-corrected p value for differential expression between the mock and infected samples (n = 2 biological replicates). Grey vertical lines indicate a transcript abundance fold change of 2. Genes which have fold changes greater than this threshold and a p ≤ 0.05 value of less than 0.05 are considered significantly differentially expressed and coloured orange if up-regulated and blue if down-regulated. Selected genes are annotated in red. (D) Volcano plot showing the relative change in translation efficiency of cellular transcripts, and the FDR-corrected p value, between the mock and infected samples (n = 2 biological replicates). Colours and fold change and p value thresholds as in C. (E) Analysis of RPFs mapping to Atf4 (NCBI RefSeq mRNA NM_009716). Cells were infected with MHV-A59 or mock-infected and harvested at 5 h p.i. (libraries from replicate 2) or 8 h p.i. RPFs are plotted at the inferred position of the ribosomal P site and coloured according to phase (which position within the codon the 5′end of the read maps to: pink for 0, blue for +1, yellow for +2). The main ORF (0 frame) is shown at the top in pink, with start and stop codons in all three frames marked by green and red bars (respectively) in the three panels below. The two yellow rectangles in the +2 frame indicate the known Atf4 uORFs (the first of which is only three codons in length). Dotted lines serve as markers for the start and end of the features in their matching colour. Note that read densities are plotted as reads per million host-mRNA-mapping reads, and that bar widths were increased to 12 nt to aid visibility, and therefore overlap, and were plotted sequentially starting from the 5′ end of the transcript. (F) Plot of log2(fold changes) of translation efficiency (TE) vs transcript abundance for all genes included in both analyses. Grey lines indicate fold changes of 2. Fold changes are plotted without filtering for significant p values. Selected genes are marked: genes up-regulated predominantly by one of either transcription or TE are marked in orange (upper middle and right middle sections), and Chop, which is up-regulated at the level of both transcription and TE, is marked in green (top right section).
Fig 2.
MHV infection and activation of the unfolded protein response.
17 Cl-1 cells were incubated in the presence of tunicamycin (2 μg/ml) or infected with MHV-A59 (MOI 5) and harvested at 2.5, 5, 8 and 10 h p.i. (A) Western blot analysis of ATF4, p-eIF2α, eIF2α, PERK and MHV N proteins. GAPDH and eIF2α were used as loading controls. Molecular masses (kDa) are indicated on the left and the p-eIF2α band is indicated by a red asterisk. Protein band quantifications for p-eIF2α, normalised by eIF2α and given relative to the timepoint-matched mock value, are provided below the immunoblot. (B) RT-qPCR of Chop and Gadd34 mRNA for three biological replicates of a timecourse of MHV infection or tunicamycin treatment. Data are normalised using Rpl19 as a housekeeping gene and presented as fold change of expression relative to mock-infected cells (marked as a dashed line). (C) Mock-infected (left upper panel) and MHV-infected (right upper panel) 17 Cl-1 cells were harvested at 5 h p.i. Cytoplasmic lysates were resolved on 10–50% sucrose density gradients. Gradients were fractionated and fractions monitored by absorbance (A254 nm). Twelve numbered fractions were collected and proteins extracted, resolved by 12% SDS-PAGE and analysed by immunoblotting using the indicated antibodies (anti-S6 as 40S marker, anti-RPL10 as 60S marker). Mock-infected (left lower panel) and MHV-infected (right lower panel) 17 Cl-1 cells were harvested at 5 h p.i. in high-salt lysis buffer (400 mM KCl) and analysed as described above. Lane "Inp" contains whole cell lysate. (D) RT-PCR analysis of Xbp1-u and Xbp1-s mRNAs. Rpl19 RT-PCR product was used as a loading control. Molecular size markers (nt) are indicated on the left. Xbp1 splicing was quantified as the ratio Xbp1-s / (Xbp1-s + Xbp1-u), and the extent of splicing relative to the timepoint-matched mock is shown below each lane. The band that migrates above Xbp1-u is thought to represent a duplex of Xbp1-s and Xbp1-u, known as the “hybrid” band (Xbp1-h) [116]. (E) RT-qPCR of Bip, Calreticulin and Grp94 mRNA for three biological replicates of a timecourse of MHV infection or tunicamycin treatment. Data are normalised as in B. (F) Cell lysates were analysed by 12% SDS-PAGE and immunoblotted using anti-BiP and anti-N antibodies. GAPDH was used as a loading control. Protein band quantifications for BiP were normalised to GAPDH. Immunoblots and agarose gels are representative of three biological replicates.
Fig 3.
Effect of UPR inhibitors on MHV replication.
(A) MHV-infected cells (MOI 5) were treated with UPR inhibitors (5 μM PERKi, 2 μM ISRIB, 60 μM IREi, or 100 μM AEBSF). The inhibitors were added to the cells immediately after the virus adsorption period and maintained in the medium until cells were harvested 8 h later. Plaque assays were performed with serial dilutions of the supernatant containing released virions from 17 Cl-1 cells infected with MHV-A59 in the presence or absence of the UPR inhibitors. Values show the mean averages of the titration of three biological replicates. Error bars represent standard errors. (B-D) MHV-infected cells (MOI 1 and MOI 5) were treated with dual combinations of the UPR inhibitors. The inhibitors were added to the cells immediately after the virus adsorption period and maintained in the medium until cells were harvested 8 h later. (B) Released virions were quantified as described in A. (C) Western blot analysis of MHV N protein. GAPDH was used as a loading control. Protein band quantifications for N protein, normalised by GAPDH and given relative to untreated/infected cells, are provided below. Immunoblots are representative of three biological replicates. (D) Representative images of mock- and MHV-infected cells at 8 h p.i. under no-drug or IREi 60 μM/AEBSF 100μM treatment conditions. All t-tests were two-tailed and did not assume equal variance for the two populations being compared (*p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). All p-values are from comparisons with the respective untreated control at the same MOI.
Fig 4.
Mechanistic analysis of UPR activation by SARS-CoV-2 proteins.
HEK-293T cells were transfected with plasmids encoding SARS-CoV-2 S (S-HA) or ORF8 (ORF8-FLAG), mock-transfected, or treated with tunicamycin (Tn). At 8 h p.t., cells were treated with 60 μM IREi and 100 μM AEBSF and then harvested at 24 and 36 h p.t. (A) Western blot analysis of ORF8-FLAG, S-HA, HERP, BiP, PERK, ATF4, p-eIF2α and ATF6 proteins. The specific p-eIF2α and ATF6-Nt bands are indicated with a red asterisk. Protein band quantifications for HERP, BiP, p-eIF2α and ATF6-Nt, normalised by eIF2α as a loading control and given relative to the mock, are provided below the respective immunoblots. (B) RT-PCR analysis of XBP1-u and XBP1-s mRNAs, performed as described in Fig 2D. Immunoblots and agarose gels are representative of three biological replicates. “h p.t.” = hours post-transfection.
Fig 5.
Induction of the UPR in SARS-CoV-2 infected cells and the effect of UPRi.
Vero CCL81 cells (A) or Calu3 cells (B) were incubated in the presence of tunicamycin (2 μg/ml) or infected with SARS-CoV-2 (MOI 1). Infected Vero CCL81 cells were treated with 60 μM IREi and 100 μM AEBSF immediately after the virus adsorption period and inhibitors were maintained in the medium until cells were harvested 24 and 48 h later. Infected Calu3 cells were harvested at 24 h p.i. (A-B) Western blot analysis (upper) of SARS-CoV-2 S, BiP, HERP, PERK, ATF4, p-eIF2α and ATF6 proteins from cell lysates of Vero CCL81 cells (A) or Calu3 (B) infected cells. The specific p-eIF2α and ATF6-Nt bands are indicated by red asterisks. Protein band quantifications, normalised by eIF2α as a loading control and given relative to the mock, are provided below the respective immunoblots. RT-PCR analysis of XBP1-u and XBP1-s mRNAs (lower), performed as described in Fig 2D. Immunoblots and agarose gels are representative of three biological replicates. (C) TCID50 assays were performed with serial dilutions of the supernatant containing released virions from Caco2 cells infected with SARS-CoV-2 (MOI 0.01) for 48 h in the presence or absence of the indicated UPRi combinations. (D) Plaque assays were performed with serial dilutions of the supernatant containing released virions from Vero CCL81 or Calu3 cells infected with SARS-CoV-2 (MOI 1 and MOI 5) for 24 h in the presence or absence of 60 μM IREi and 100 μM AEBSF. Values show the mean averages of the titration of three biological replicates. Error bars represent standard errors. All t-tests were two-tailed and did not assume equal variance for the two populations being compared (*p < 0.05, ** p < 0.01). Replicates with titres below the limit of detection (LoD) were excluded from t-tests, precluding some conditions from statistical assessment.
Fig 6.
Effect of specific inhibition of IRE1α and ATF6 pathways on SARS-CoV-2 replication.
Vero CCL81 cells were incubated in the presence of tunicamycin (2 μg/ml) or infected with SARS-CoV-2 (MOI 5) and treated with 15 μM Ceapin-A7 and 10 μM KIRA8 as individual treatments or in combination. The inhibitors were added to the cells immediately after the virus adsorption period and maintained in the medium for 24 or 48 h. (A) Western blot analysis (upper) of SARS-CoV-2 S, PERK, ATF4, p-eIF2α and ATF6. The specific p-eIF2α and ATF6-Nt bands are indicated by red asterisks. Protein band quantifications, normalised by eIF2α as a loading control and given relative to the mock, are provided below the respective immunoblots. RT-PCR analysis of XBP1-u and XBP1-s mRNAs (lower), performed as described in Fig 2D. Immunoblots and agarose gels are representative of three biological replicates. (B) Plaque assays were performed with serial dilutions of the supernatant containing released virions at 24 and 48 h p.i. Values show the mean averages of the titration of four biological replicates. Error bars represent standard errors. All p-values are from comparisons with the respective untreated control, with *p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. (C) RT-qPCR of vRNA from two biological replicates of Vero CCL81 cells infected and treated as described above. Data are normalised as described in Fig 2B.