PMC

MNV infection in macrophages induces cytokine transcription but inhibits their secretion. (A) RAW 264.7 cells were either MNV infected (MOI of 5) for 9, 12, and 15 h or poly(I⋅C) treated. RNA samples were taken and analyzed via RT-qPCR for the cytokines IFN-β (i), TNF-α (ii), and IL-6 (iii). (B) RAW 264.7 cells were mock infected, infected with MNV (MOI of 5) for 9, 12, and 15 h, or poly(I⋅C) or poly(I⋅C)+BFA treated. Cell culture supernatants were analyzed for the secretion of the cytokines IFN-β (i), TNF-α (ii), and IL-6 (iii) by using ELISA (n = 3, ANOVA, means ± the SEM; **, P < 0.01; ****, P < 0.0001).

Model. During MNV infection, viral factors such as proteins and/or RNA (blue hexagon) phosphorylate eIF2α (green oval) via PKR (orange rectangle), as well as stalling translation initiation by the MNV NS3 protein. However, this translational arrest is uncoupled from the PKR–p-eIF2α axis. These stalled preinitiation complexes typically aggregate with G3BP1 (gray oval) and form SGs (red cloud). However, MNV viral factors sequester G3BP1 to the MNV RC (yellow circle) to promote replication. This allows the inhibition of cap-dependent host cell translation, as well as inhibiting the formation of SGs.

Expression of MNV NS3 protein abolishes host cellular translation. (A) HEK 293T cells were transfected with cDNA expression plasmids encoding the individual His-tagged MNV NS proteins for 18 h, at which point the cells were pulsed with puromycin as previously described, and whole-cell lysates were obtained and immunolabeled with anti-puromycin antibodies. (B) Densitometry analysis of puromycin signal in MNV NS protein-transfected cells compared to mock-transfected cells (n = 3, ANOVA, means ± the SEM; **, P < 0.01; ****, P < 0.0001). (C) Vero cells were transfected with single His-tagged MNV NS proteins and treated with puromycin before the cells were fixed and permeabilized for IF. Cells were immunolabeled with antibodies against puromycin (green), 6×His (red), and DAPI. Samples were captured by using a Zeiss LSM 710 confocal microscope and analyzed with ZEN software.

Treatment with PKR inhibitor C16 abolishes phosphorylation of eIF2α but does not rescue host translation. (A) RAW 264.7 cells were either uninfected or MNV infected (MOI of 5), treated with either DMSO, C16 (1 μM), ISRIB (0.5 μM), or C16+ISRIB at 1 hpi for 12 h before cell lysate samples were obtained. Lysates were analyzed via immunoblotting and immunolabeled with anti-NS7, anti-p-eIF2α, or anti-actin antibodies. (B) Quantification of p-eIF2α. Ratios: uninfected DMSO/C16 (1:0.64 ± 0.12, P = 0.015, n = 5), DMSO/ISRIB (1:0.72 ± 0.07, P = 0.0013, n = 3), and DMSO/C16+ISRIB (1:0.96 ± 0.23, P = 0.83, n = 3); MNV-infected DMSO/C16 (0.54 ± 0.09, P = 0.0008, n = 5), DMSO/ISRIB (0.88 ± 0.06, P = 0.03, n = 3, and DMSO/C16+ISRIB (1:0.70 ± 0.13, P = 0.02, n = 3). Statistics were calculated by using a Student t test; the means ± the SEM are shown. (C) RAW 264.7 were either uninfected or MNV infected (MOI of 5) and treated with either DMSO or C16 (1 μM) at 1 hpi for 12 or 15 h. At 30 min before the cell lysate samples were obtained, the cells were treated with puromycin (10 μg/ml for 30 min) and immunolabeled with anti-NS7, anti-p-eIF2α, anti-puromycin, and anti-calnexin antibodies.

MNV recruits G3BP1 and requires G3BP1 for efficient viral replication. (A) BMMs were mock infected (a to d), NaAs treated (250 μM for 20 min) (e to h), infected with MNV (MOI of 5) for 12 h (i to l), or MNV infected and NaAs treated (m to p). Cells were fixed for IF analysis and immunolabeled with anti-eIF3η (magenta), anti-G3BP1 (green), VPg (red, indicating infection), and counterstained with DAPI (blue). (B) BMM cells were mock infected (a to d) or MNV infected (MOI of 5, 12 h) (e to h). Cells were fixed for IF analysis and immunolabeled with anti-G3BP1 (green) or anti-NS4 (red) and counterstained with DAPI (blue). (A and B) Samples were captured via the Zeiss LSM 710 confocal microscope and analyzed with ZEN software. (C and D) BMMs were untreated, siControl or siG3BP1 treated, and either mock infected or MNV infected (MOI of 5). (C) Whole-cell lysates were collected for WB (immunolabeled with anti-NS7, anti-G3BP1, and anti-GAPDH). (D) Tissue culture fluids were collected for plaque assay. n = 3 (means ± the SEM are shown, and an unpaired two-tailed t test was performed). (E) Schematic demonstrating WT-mG3BP1, mG3BP1-ΔRGG, and mG3BP1-ΔRRMRGG protein mutant constructs. Each construct was transfected into BV2, and the expression levels were demonstrated by WB. (F) WT-BV2 cells and BV2 -mG3BP1-KO cells were transfected with WT-mG3BP1, mG3BP1-ΔRGG, mG3BP1-ΔRRMRGG, and an empty vector. After transfection, the cells were infected with MNV, and at 12 and 24 hpi tissue culture fluids were collected for plaque assay. n = 3 (the means ± the SEM are shown, and an unpaired two-tailed t test was performed).

MNV infection phosphorylates eIF2α and shuts down host cell translation. (A) BMMs were uninfected, uninfected but NaAs treated (250 μM for 20 min), or MNV infected (MOI of 5) for 12 h. The WB was immunolabeled with anti-NS7, anti-p-eIF2α, and anti-eIF2α antibodies. Nonspecific lanes were removed to generate the image. (B) Immunoblot analysis of uninfected, uninfected/NaAs-treated (250 μM for 20 min), or MNV-infected (MOI of 5) cell lysates harvested at 3, 6, 9, and 12 hpi. The WB was immunolabeled with anti-NS7, anti-p-eIF2α and anti-actin antibodies. (C and D) BMM cells were either infected with MNV (MOI of 5) or left uninfected and analyzed for their translation using puromycin (10 μg/ml). (C) Immunoblot analysis of puromycin-treated (20 min) cell lysates harvested at 3, 6, 9, 12, and 15 hpi. The WB was immunolabeled with anti-puromycin, anti-NS7, anti-p-eIF2α, and anti-actin antibodies. (D) IF analysis of puromycin-treated (10 μg/ml for 30 min) cells at 6, 9, and 12 h postinfection. Cells were stained with anti-puromycin, anti-NS5, and DAPI for the merged image. Asterisks indicate uninfected cells displaying a high signal for anti-puromycin. Samples were analyzed via the Zeiss LSM 710 confocal microscope and analyzed with ZEN software. (E) Quantification of puromycin raw integrated density (RawIntDen) level of mock-infected and MNV-infected cells at 6, 9, and 12 hpi. 6 hpi, mock, n = 113, bystander, n = 108, and MNV, n = 38; 9 hpi, mock, n = 125, bystander, n = 108, and MNV, n = 40; 12 hpi, mock, n = 260, bystander, n = 148, and MNV, n = 81. Means ± the SEM are shown, and an unpaired two-tailed t test was performed. ****, P < 0.0001.

MNV infection alters SG formation in sodium arsenite-treated cells without affecting key SG protein levels. (A) BMMs were mock infected (a to c), infected with MNV (MOI of 5) for 12 h (d to f), NaAs treated (250 μM for 20 min) (g to i), or MNV infected and NaAs treated (j to l). The cells were fixed for IF analysis and immunolabeled with eIF3η (green), VPg (red, indicating infection), and DAPI (blue). Samples were captured via the Zeiss LSM 710 confocal microscope and analyzed with ZEN software. (B and C) SGs in NaAs-treated mock-infected (218 cells) and MNV-infected (182 cells) samples were counted from two independent experiments each and collated. (B) Box-and-whiskers plot, where the whiskers represent minimum to maximum, and the box represents the mean with error bars ± the SEM. An unpaired two-tailed t test was performed (****, P < 0.0001). (C) Quantitation demonstrating the total number of cells (y axis) containing various amount of SGs (x axis). The blue line represents the number of SGs in uninfected cells (n = 218 cells), and the red line represents the number of SGs in MNV-infected cells (n = 182 cells). (D) BMM cells were either mock infected or MNV infected (MOI of 5), and whole-cell lysates were collected at 3, 6, 9, and 12 hpi. A Western blot was immunolabeled with anti-eIF3η, anti-G3BP1, anti-TIA-1, anti-NS7, and anti-actin antibodies.