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.