Table 1

Software tools for predicting structure and functions of the ASFV MGF110-5L-6L"

Fig. 1

The phosphorylation level of eIF2α was significantly up-regulated by the ectopic expression of MGF110-5L-6L A: Illustration of ATF4-RLuc reporter (top). HEK293T cells were cotransfected with ATF4-RLuc reporter and an empty vector or a vector expressing MGF110-5L-6L-Flag. At 24 h posttransfection, the cells were lysed and subjected to RLuc assays. A positive control for eIF2α phosphorylation was set by thapsigargin (TG, 1 μmol·L-1) treatment for 6 h on cells transfected with ATF4-RLuc; B: PK-15 cells were cotransfected with ATF4-EGFP reporter and FLAG vector or MGF110-5L-6L-Flag. At 24 h posttransfection, the cells were subjected to fluorescence analysis. TG was used as positive control; C and D: PK-15 and 3D4/21 cells were transfected with a MGF110-5L-6L-Flag expressing vector with an increasing dose or an empty Flag vector for 24 h. The cells were then subjected to Western blot analysis (left). The grayscale values of the protein bands were analyzed by Image J (right). Data were presented as the means ± SD of three independent experiments. **: P<0.01, n.s.:not significant"

Fig. 2

Ectopic expression of MGF110-5L-6L induces host translation shutoff A: Schematic diagrams showing the role of phosphorylation of eIF2α in host translational control; B: PK-15 cells transfected with the FLAG empty vector or FLAG-tagged MGF110-5L-6L for 24 h were incubated with puromycin (5 μg·mL-1) at 37℃ for 30 min. As a positive control, cells transfected with an empty vector were treated with 0.5 mmol·L-1 Ars for 45 min before addition of puromycin. The cells were then fixed, permeabilized, and processed for IFA. A FLAG-specific monoclonal antibody was used to detect MGF110-5L-6L(green). Puromycylated chains are visualized using an antipuromycin antibody (red). Nuclei were stained with DAPI (blue). Scale bar, 20 μm; C: PK-15 cells transfected with the MGF110-5L-6L-Flag expressing vector with an increasing dose or an empty Flag vector for 24 h were induced with puromycin for 30 min. The cells were then subjected to Western blot analysis (left). The grayscale values of the protein bands were analyzed by Image J (right); D: 3D4/21 cells were treated with 0.5 mmol·L-1 Ars for 45 min, or transfected with the FLAG empty vector or FLAG-tagged MGF110-5L-6L for 24 h, with (+ISRIB) or without (-ISRIB) 0.5 μmol·L-1 of ISRIB. At 30 min before the cell lysate samples were obtained, the cells were incubated with 5 μg·mL-1 of puromycin to label the nascent polypeptidic chains. Data were the means ± SD of three independent experiments. **: P<0.01"

Fig. 3

Ectopic expression of MGF110-5L-6L induces stress granule (SG) formation A: A schematic diagram showing the relationship of eIF2α phosphorylation, translation arrest, and SG formation; B: PK-15 cells were transfected with an empty Flag vector or a MGF110-5L-6L-Flag expressing vector for 24 h, with or without 0.5 μmol·L-1 of ISRIB; As a control, cells transfected with FLAG vector or DP71L-FLAG for 24 h were treated with 0.5 mmol·L-1 Ars for 45 min. The cells were then fixed, permeabilized, and incubated with anti-G3BP1 and anti-FLAG antibodies and then with secondary antibodies conjugated with AF488 (green) and AF594 (red), respectively. Nuclei were stained with DAPI (blue), and localization was determined using confocal microscopy. Scale bar, 20 μm; C: Representative bar plot of the percentage of cells displaying SGs. Total of 100 cells in each group were counted in each experiment and the error bars represented mean ± SD (n = 3). **: P<0.01"

Fig. 4

Sequence alignment, structure and function analysis of MGF110-5L-6L protein A: Multiple sequence alignment of the indicated ASFV isolates of viral protein MGF110-5L-6L. Predicted secondary structure of MGF110-5L-6L was shown and the cysteine-rich domains were highlighted. Red arrow indicated the first amino acid of the MGF110-6L from African virus isolates; B: Predicted signal peptide and cleavage sites of MGF110-5L-6L; C: Visualization of the predicted tertiary structure of MGF110-5L-6L"

Fig. 5

Confocal immunofluorescence analysis of the intracellular distribution of the MGF110-5L-6L protein PK-15 cells were cotransfected with an empty Flag vector or a MGF110-5L-6L-Flag expressing vector and one of the following organelle markers: pDsRed2-ER, pDsRed2-Golgi, pDsRed2-Mito, pDsRed2-LAMP, or pDsRed2-Peroxi. At 24 h posttransfection, the cells were fixed and incubated with anti-FLAG antibody and then with secondary antibody conjugated with AF488 (green). Nuclei were counterstained with DAPI (blue). The organelle marker was directly visualized (red), and localization was determined using confocal microscopy. The overlapping coefficient (R) was shown in merged images, and intensity profile of linear region of interest (ROI) across the PK-15 cell contained with MGF110-5L-6 and other organelle markers. Scale bar, 20 μm"

Fig. 6

Ectopic expression of MGF110-5L-6L enhances eIF2α phosphorylation by up-regulation of PERK and PKR activity A: Diagrams of four major stress pathways leading to eIF2α phosphorylation by different kinases, PERK, PKR, GCN2, and HRI; B and C: 3D4/21 and PK-15 cells transfected with the MGF110-5L-6L-Flag expressing vector with an increasing dose, or an empty Flag vector for 24 h were subjected to Western blot analysis. As a positive control, cells were treated with TG (1 μmol·L-1) for 6 h; D and E: The grayscale values of the protein bands for panel B and C were analyzed by Image J; F and G: 3D4/21 cells were transfected with an empty Flag vector or a MGF110-5L-6L-Flag expressing vector, with or without GSK2606414 (10 μmol·L-1) or C16 (1 μmol·L-1) as indicated for 24 h. Lysates were analyzed via immunoblotting. Data were the means ± SD of results of three independent experiments. **, P<0.01, n.s., not significant"