Fig 2: Location of mutations of eIF2Bα on its structure. a Structure of the eIF2Bα dimer showing the location of the mutated residues. All the VWM associated mutations affect sites in the α-helix and β-sheet rich Rossmann-like fold rather than the α-helical bundle furthest from the interaction interface of the homodimer. b The effect of the Asn208Tyr mutation on the proposed phospho-eIF2 binding pocket. The ribbon and spacefill structures show the location of the pocket on the structure of a single monomer of the subunit. The right hand panels show the WT (Asn208) residue relative to the sulphate ion mimicking a phosphate group and a co-ordinating water molecule (upper panel), and the predicted change to the pocket following in silico mutation of Asn208 to Tyr

Fig 3: Gene silencing by siRNA delivered by nanoparticles in PEMs. (A) WB analysis of the silencing of PERK and its downstream effector phospho–eiF2–α in PEMs treated with LIPO/siPERK and PDAMNPs/siPERK for 48 h. The ER stress inducer tunicamycin (TM, 2 µg/mL) was used as a control along with PDA–MNPs alone. (B) Densitometric analysis of protein bands using the protein expression of α-Tubulin as an internal control. (C,D) Histograms showing mRNA levels of phospho–eiF2–α downstream effectors (ATF4 and CHOP) and ER stress sensors (ATF6 and XBP1) detected by q–RT–PCR following the silencing of PERK in PEM cells with PDA-MNPs/siPERK using LIPO/siPERK as a positive control for gene silencing. Gene expression was expressed as a fold change relative to untreated cells. The data are expressed as the mean ± SD of three independent experiments using one-way ANOVA with the Bonferroni comparison post-test vs. the untreated controls (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p < 0.0001).

Fig 4: FMDV infection induces autophagy through the EIF2S1-ATF4-AKT-MTOR cascade. (A) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. The phosphorylation of AKT, AMPK, MTOR and ULK1 were analyzed by western blot. ACTB was used as a sample loading control. (B) PK-15 cells were mock infected or infected with FMDV (MOI = 1) for 1.5, 2 and 3 h. ATF4 and phosphorylation of EIF2S1 were analyzed by western blot. (C) ATF4 KD and wild-type cells cells were infected with FMDV (MOI = 1). ATF4, LC3B and phosphorylation of AKT, MTOR and ULK1 were analyzed by western blot. (D) ATF4 and scrambled knockdown cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by FMDV infection (MOI = 1) for 3 h. The fluorescence signals were visualized by confocal immunofluorescence microscopy. Scale bars: 10 μm. (E and F) ATF4 KD and wil-type cells cells were infected with FMDV (MOI = 1) for 3 h. At 3 hpi, both the extracellular and intracellular copy numbers of FMDV were measured by qRT-PCR; both the extracellular and intracellular virus titers were measured by TCID50. The data represent the mean ± SD of 3 independent experiments. **P < 0.01, ***P < 0.001.

Fig 5: Effect of SFSV NSs on mRNA translation. (a and b) Influence of SFSV NSs on a bicistronic reporter system with an upstream canonical (i.e., eIF-dependent) firefly luciferase ORF and a downstream Renilla luciferase ORF initiated from the eIF-independent IRESCrPV. HEK293 cells were transfected with the bicistronic reporter plasmid and expression plasmids for SFSV NSs containing either a C- or N-terminal 3×FLAG tag (SFSV NSs-3×FLAG and 3×FLAG-NSs SFSV, respectively) or for an irrelevant negative control (3×FLAG-ΔMx). Lysates were assayed for eIF2-dependent firefly (Fluc) (a) and IRES-dependent Renilla luciferase (Rluc) (b) activities (arbitrary units [AU]). Shown are data from 1 of 3 independent experiments. Each of these was conducted with three technical (i.e., parallel) replicates. Bars show means ± SDs. ****, P ≤ 0.0001; ***, P < 0.001 (one-way ANOVA, corrected for multiple comparisons using Dunnett test). For the Rluc data, none of the tests reached the significance level. (c) C- and N-terminally tagged SFSV NSs (SFSV NSs-3×FLAG and 3×FLAG-NSs SFSV, respectively) were transiently expressed in HEK293 cells, and expression levels were analyzed by immunoblotting (representative of 3 experiments). (d) A549 cells were infected with SFSV, MP12, or clone 13 (MOI, 1), and currently translated proteins were labeled by the addition of puromycin 12 h postinfection, followed by subsequent immunoblot analysis (representative of 4 experiments). Staining of viral N proteins served as marker for infection. Data are representative and from 1 of 3 independent experiments each with 3 technical replicates (a and b), from 1 of 3 independent experiments (c), or from 1 of 4 independent experiments (d).