Fig 2: PKR-mediated shutdown of translation and antagonistic strategies employed by viral proteins. (a) Shutdown of translation initiation mediated by PKR: in response to recognition of double-stranded RNA (dsRNA), PKR phosphorylates eIF2aSer51, resulting in nonproductive binding of the latter to eIF2B and, consequently, global inhibition of translation. Due to limiting concentrations of eIF2B compared to that of eIF2, partial eIF2a phosphorylation is sufficient to shutdown protein synthesis. (b) Previously reported viral strategies for PKR antagonism: viral proteins (blue) sequester viral RNA or affect PKR levels (such as NSs proteins of phleboviruses RVFV and TOSV), PKR activation, or the phosphorylation state of eIF2a. (c) Mechanism used by SFSV NSs: while PKR activation, PKR phosphorylation, and eIF2a phosphorylation occur in response to the virus infection and even the binding of p-eIF2a to eIF2B occurs, SFSV NSs enables cap-dependent translation by targeting eIF2B in a way that somehow neutralizes the nonproductive mode of eIF2B imposed by p-eIF2a binding.

Fig 3: Biochemical effects of mutations of eIF2Ba on eIF2B complex formation. a HEK293 cells were transfected with plasmids encoding myc-tagged eIF2B subunits andHis6-myc-tagged eIF2Ba. ‘No His-tag’ indicates that a vector encoding myc-tagged eIF2Ba was used instead. Lysates from transfected cells were analysed by western blotting for the myc tag to verify even expression of each subunit. An actin loading control is included. b Lysates were subjected to affinity purification using Ni2+ agarose and the bound material was analysed by western blot for myc tag to test for the presence of other subunits associated with His6-mys-eIF2Ba and phospho- and total eIF2a to assess substrate binding. c The myc-signal for the WT and indicated mutations were quantified and normalised to the level of eIF2Ba. Data are shown relative to the level of the WT pulldown. Data are shown as the relative expression to WT ± SEM of 3 independent experiments. P-values are as indicated on the figure. d Levels of total and phospho-eIF2 from pulldowns were quantified and normalised to the level of eIF2Ba

Fig 4: FMDV capsid protein VP2 induced autophagy. (A) PK-15 cells were transfected with empty vectors or various plasmids expressing FLAG-tagged VP1, VP2and VP3 proteins for 24 h. LC3B and FMDV capsid proteins were analyzed by western blot. ACTB was used as a sample loading control. (B) Cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. Cells were fixed and analyzed by immunofluorescence using anti-LC3B antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 µm. (C) PK-15 cells were transfected with empty vectors or pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of EIF2S1, AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control. (D) ATF4 and scrambled knockdown cells were transfected with pCMV-Flag-VP2 for 24 h. LC3B and phosphorylation of AKT and MTOR were analyzed by western blot. ACTB was used as a sample loading control.

Fig 5: Absence of UPR and ISR activation in the liver of mistranslating A226Y mice. (a) Western blots showing total and specific phosphorylation levels for key players in the three branches of the ER-UPR. Significance level for comparison between A226Y and WT (N = 5): p-IRE1 (Ser724)/IRE1a p = 0.43; p-eIF2a (Ser51)/eIF2a p = 0.55; GADD34 p = 0.77; ATF6 p = 0.48, BiP p = 0.64. Densitometry values were normalized to tubulin as the loading control. (b) qRT-PCR quantification of spliced XBP1 relative to un-spliced XBP1 (N = 4, p = 0.94). (c) Western blot of total ATF4 protein normalized to tubulin (N = 5, p = 0.01). (d) Western blot of total Bim protein normalized to tubulin (N = 5, p = 0.45). Plots show mean, SEM, and individual data points. * p < 0.05, calculated by unpaired two-sided Student’s t-test. N = number of independent mice in each comparison. Full gel images are provided in the Supplementary Materials.