Journal: Virology Journal

Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

doi: 10.1186/s12985-025-02799-3

Figure Lengend Snippet: Inhibition of MNK1 phosphorylation in GGNNV-infected GB cells reduces p-MNK1 and p-eIF4E production as well as coat protein translation. A GB cells were infected with GGNNV (MOI = 100). Cells were fixed at 0, 12 and 24 hpi and immunostained for p-MNK1 (green) and coat protein (red). B The cytotoxic effect of MNK1 phosphorylation inhibitor, CGP57380 on GB cells was evaluated by MTT assay. GB cells cultured in a 96-well plate were treated with different concentrations of CGP57380 for 24 h. The cell viabilities were detected with a MTT kit. Values are presented as mean ± SD ( n = 3). C Immunocytochemical staining of p-MNK1 (green) and coat protein (red) in DMSO treated (control) GGNNV-infected GB cells. D Immunocytochemical staining of p-MNK1 (green) and coat protein (red) in 10 µM CGP57380 treated GGNNV-infected GB cells. E Relative coat protein expressions (%) (co-stained with p-MNK1) in DMSO and CGP57380 treated cells were analyzed at 6, 12, 18 and 24 hpi ( n = 54 to 66 cells). F Relative fluorescence intensity of p-MNK1 in DMSO and CGP57380 treated GGNNV-infected cells ( n = 50 to 60 cells). G Relative fluorescence intensity of p-eIF4E in DMSO and CGP57380 treated GGNNV-infected cells ( n = 50 to 60 cells). DMSO and inhibitor treated GB cells were infected with GGNNV (MOI = 100). The samples were collected and fixed at 0, 6, 12, 18 and 24 hpi and then proceeded with immunocytochemical staining using anti-p-eIF4E and anti-RG-M18 antibodies (image is shown in supplementary Fig. ). The mean ± SD for coat protein, p-MNK1 and p-eIF4E were plotted. ns, not significant; **, P < 0.002; ****, P < 0.0001 (two-way ANOVA test). The nuclei (blue) were stained with DAPI. Scale bar = 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; GB, grouper brain; GGNNV, giant grouper nervous necrosis virus; hpi, hour post infection; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SD, standard deviation

Article Snippet: Rabbit anti-phospho-eIF4E (p-eIF4E) (Ser209) polyclonal antibody (#9741), rabbit anti-phospho-eIF4E-BP1 (p-eIF4E-BP) (Ser65) monoclonal antibody (D9G1Q) (#13443), rabbit anti-phospho-MNK1 (p-MNK1) (Thr197/202) polyclonal antibody (#2111), rabbit anti-phospho-p44/42 MAPK (p-ERK) (Thr202/204) monoclonal antibody (D13.14.4E) (#4370), rabbit anti-phospho-p38 MAPK (p-p38) (Thr180/Tyr182) monoclonal antibody (D3F9) (#4511), rabbit anti-phospho-S6 ribosomal protein (p-RPS6) (Ser235/236) monoclonal antibody (D57.2.2E) (#4858) and rabbit anti-phospho-p70S6 kinase (p-p70S6K) (Thr389) monoclonal antibody (#9205) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Phospho-proteomics, Infection, MTT Assay, Cell Culture, Staining, Control, Fluorescence, Virus, Standard Deviation

Journal: Virology Journal

Article Title: Translation of nervous necrosis virus involves eIF4E but not RPS6 phosphorylation and viral particle assembly in remodeled microtubule-organizing center

doi: 10.1186/s12985-025-02799-3

Figure Lengend Snippet: Schematic illustration of how NNV hijacks host machinery for virus protein synthesis and particle assembly. After several rounds of RNA replication/transcription near the mitochondria outer membrane, NNV RNAs are transported from mitochondrial spherules (site of replication) to translation factories. These factories are fused together and later concentrated in perinuclear area at a reorganized MTOC. The resulting viral factories (VFs) are architecturally supported by cytoskeleton proteins. Nuclear movement and reshaping may occur due to LINC complexes connecting nucleus to cytoskeletal elements. The VFs and remodeled MTOC act as a microenvironment to divert host proteins like p-p38, p-ERK, p-MNK1, p-eIF4E, p-eIF4E-BP, RPS6 and other translation factors important for NNV coat protein translation. Outside the VFs and remodeled MTOC, NNV downregulates p-p70S6k/p-RPS6 pathway which phosphorylates RPS6 crucial for host translation. Moreover, NNV inhibits host translation by inducing translocalization and sequestration of PABP in nucleus. These events are followed by degradation of PABP via the 26 S proteasome system . LINC, linker of nucleoskeleton and cytoskeleton; MTOC, microtubule-organizing center; PABP, poly(A) binding protein; VF, Viral Factory. Image created with Biorendor.com

Article Snippet: Rabbit anti-phospho-eIF4E (p-eIF4E) (Ser209) polyclonal antibody (#9741), rabbit anti-phospho-eIF4E-BP1 (p-eIF4E-BP) (Ser65) monoclonal antibody (D9G1Q) (#13443), rabbit anti-phospho-MNK1 (p-MNK1) (Thr197/202) polyclonal antibody (#2111), rabbit anti-phospho-p44/42 MAPK (p-ERK) (Thr202/204) monoclonal antibody (D13.14.4E) (#4370), rabbit anti-phospho-p38 MAPK (p-p38) (Thr180/Tyr182) monoclonal antibody (D3F9) (#4511), rabbit anti-phospho-S6 ribosomal protein (p-RPS6) (Ser235/236) monoclonal antibody (D57.2.2E) (#4858) and rabbit anti-phospho-p70S6 kinase (p-p70S6K) (Thr389) monoclonal antibody (#9205) were purchased from Cell Signaling Technology.

Techniques: Virus, Membrane, Binding Assay