Fig 2: EDF1 recruits GIGYF2•EIF4E2 to collided ribosomes.(A) (Left) UV (A258) absorbance across 10–50% sucrose gradients from lysates of HEK293 Flp-In TREx WT and ?EDF1 cells left untreated (UT, blue trace) or treated with 1.8 µM emetine (EL, orange trace) for 15 min; fractions 6–8 pooled for light polysomes; fractions 9–11 pooled for heavy polysomes (n = 4). (Right) Schematic of polysome proteomics pipeline to monitor relative change in protein intensity in response to emetine treatment in light and heavy polysomes between WT and ?EDF1 cells. (B, C) Volcano plot of log2 indicated ratio (x-axis) against -log10(p-value) (y-axis) for light (B) or heavy (B) polysomes. (D) UV (A258) absorbance across 10–50% sucrose gradients from lysates of HEK293-Flp-In TREx WT and ?EDF1 cells left untreated (UT, blue trace) or treated with 1.8 µM emetine (EL, orange trace) for 15 min; TCA precipitated proteins from individual fractions were resolved by SDS-PAGE and analyzed by immunoblotting using GIGYF2 and uS3 antibodies. (n = 2) (E) Relative GFP intensity from HEK293 Flp-In TREx WT, ?EDF1, ?ZNF598, and ?EDF1?ZNF598 cells transfected with the GFP-(KAAA)20-RFP stalling reporter without (white bars, non-targeting siRNA, SCRi) or with siRNA-mediated depletion of GIGYF2 and EIF4E2 (green bars; GIGYF2i•EIF4E2i). Error bars denote SD for n = 3. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons. See also Figure 6—figure supplement 1, Figure 6—figure supplement 2 and Figure 6—source data 1.Figure 6—source data 1.Related to Figure 6B-C and Figure 6—figure supplement 1; Pooled sucrose gradient fractions (light and heavy polysomes) analysis with or without low-dose emetine treatment (1.8 µM, 15 min).16plex TMT-MS3 analysis of HEK293 Flp-In T-REx cells (WT vs ?EDF1).

Fig 3: PRRSV infection promotes RPS3 expression and nuclear accumulation in PAMs. (a) PRRSV infection increases RPS3 expression. The pulmonary alveolar macrophages (PAMs) from PBS or PRRSV HuN4-inoculated pigs were subjected to IB. (b) PRRSV infection facilitates the nuclear accumulation of RPS3. PAMs were subjected to subcellular fractionation, followed by IB of RPS3. The same blot was probed with antibodies against tubulin and histone H2A as controls for loading and fractionation. (c) Densitometry analysis of the digital blotting images in (b). The RPS3 band intensity of each fraction is shown as the relative percentage of the summed density of both corresponding cytoplasmic and nuclear fractions after normalization with tubulin and histone for cytoplasmic and nuclear fractions, respectively. Significant differences between the control group and the PRRSV-infected group are denoted by “*” for p < 0.05.

Fig 4: PRRSV increases HMGB1 binding to RPS3 in MARC-145 cells. MARC-145 cells were infected with HuN4 at an MOI of 1 for 6 h (a) and 24 h (b). The cells were harvested for IP with antibodies against HMGB1 or RPS3, followed by IB. Relative levels of RPS3 and HMGB1 are shown as folds below the images after normalization with HMGB1 or RPS3, respectively, in a densitometry analysis. The input was also included in IB. PRRSV+ pig serum was used for detecting PRRSV infection. (c) Immunofluorescence assay (IFA) shows the co-localization of HMGB1 and RPS3. MARC-145 cells were infected with HuN4 at an MOI of 1 and, 24 h later, were fixed for IFA with antibodies against HMGB1 (red) and RPS3 (green). DAPI staining of nuclear DNA is also shown (blue). Arrow points to the cell showing HMGB1-RPS3 co-localization. Bar in images denotes 25 μm.

Fig 5: Structural analysis of ribosome-bound EDF1 and Mbf1.(A) Overview of EM map and models of the 40S subunit of human non-rotated EDF1-bound ribosome. Selected r-proteins and EDF1 (orange) are shown as models in the EDF1•80S map (PDB: 6ZVH). (B) Overview of Mbf1 (violet red) bound to the yeast rotated ribosome with hybrid tRNAs. (PDB: 6ZVI). (C) Alignment of EDF1 and Mbf1 sequences colored by conservation and domain architecture of EDF1. (D) Overall structure of ribosome-bound EDF1 and Mbf1 showing a highly similar fold and binding mode with the C-terminus sandwiched between helix 16 (h16) and helix 33 (h33) of the 18S rRNA and the r-protein uS3 close to the mRNA entry channel, and the N-terminus forming a helix at the base of helix 16. (E) EDF1 and Mbf1 interact with rRNA helix 18 (h18), displacing the C-terminus of eS30. Binding of EDF1 and Mbf1 shifts helix 16 towards the ribosome, resulting in a clash of the canonical eS30 position with the new position of helix 16. See also Figure 3—figure supplement 1, Figure 3—figure supplement 2 and Figure 3—source data 1.Figure 3—source data 1.Cryo-EM data collection, refinement and validation statistics.