Interaction of Nucleolin with the Fusion Protein of Avian Metapneumovirus Subgroup C Contributes to Viral Replication

Figures

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  Figure 1. aMPV/C infection-induced cytoplasmic re-localization of NCL from the nucleus. Vero cells were infected or not with aMPV/C, and samples were harvested at 12, 24, 36, and 48 h following infection or non-infection. (A) aMPV/C RNA loads are displayed according to the number of hours following infection with aMPV/C, represented in copies/mL. (B) qRT-PCR analysis was used to identify relative quantities of NCL RNA in different samples obtained at the indicated time. (C) Western blot analysis was utilized to determine the total level of NCL expression, as well as the cytoplasmic and nuclear abundance of different samples obtained at the specified times. H3, β-actin, or β-tublin was used as an internal control. All data from three different experiments were shown as mean SD. ** p < 0.01, *** p < 0.001 were considered statistically significant; ns, not significant.

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  Figure 2. NCL promotes aMPV/C replication. (A,B) 20 μM of NCL−siRNA or increased amounts of pCMV−HA−N−NCL plasmids were transfected or mock−transfected into Vero cells. At 24 h, NCL proteins were detected by western blot. β−actin served as an internal control. (C) qRT−PCR was performed to determine the relative aMPV/C RNA level in Vero cells under both knockdown and overexpression of NCL, and western blot * p < 0.05 were considered statistically significant; ns, not significant. (D) was used to quantify the viral protein expression level under these conditions. The expression of total GAPDH protein and GAPDH mRNA were utilized as controls. (E) Vero cells transfected with NCL−siRNA or CMV−HA−N−NCL plasmids were infected with aMPV/C for 48 h, the cells were then directly observed under an immunofluorescence microscope and a specific antibody to the aMPV/C (green), and nuclei were stained with DAPI (blue).

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  Figure 3. Knocking−out NCL limits the aMPV/C replication. (A) The genomic structure of chicken NCL gene and the knock−out strategy for NCL knockout using CRISPR/Cas9. NCL−KO cells and wild−type (WT) cells were infected with aMPV/C, and the cells and supernatants were collected 48 h later. Western blotting, sequence analysis, and TCID50 assay were used to evaluate the expression of NCL and aMPV/C N (B), DNA sequence (C), and the viral titer (D). NCL−KO cells and WT cells transfected with pCMV−HA−NCL plasmids were infected with aMPV/C, and the cells were harvested 48 h later for analysis of the relative NCL RNA level (E) and of the viral protein expression level (F) using qRT−PCR and western blotting. β−Actin or GAPDH was used as an internal control.

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  Figure 4. aMPV/C F protein interacts with NCL. (A) Vero cells were transfected or not transfected with the pCMV−HA−F plasmids for 36 h and an IP assay on cell lysates was performed using HA mAb, followed by immunoblotting with mAbs against HA and NCL. (B) The plasmids of pCMV−HA−NCL and pEGFP−C1−F were co−transfected into HEK293 cells for 36 h, a Co−IP assay was subsequently performed using GFP mAb, followed by immunoblotting with mAbs against HA and GFP. (C) Vero cells transfected or not transfected with pCMV−HA−F were incubated with NCL antibody (green), HA antibody (red), and DAPI (blue), and then observed by confocal microscopy. Scale bar, 200 μm.

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  Figure 5. NCL binding aptamer AS1411 inhibits viral replication by disrupting the interaction between NCL and aMPV/C F. After 72 h of treatment with FAM−AS1411 in DF−1 cells and Vero cells, the FAM−signal and the toxicity of AS1411 were tested by confocal microscopy (A) and the CCK−8 assay (B). Vero cells and DF−1 cells treated with AS1411 or CRO for 24 h were infected with aMPV/C, and the cells and supernatants were collected at 48 h after infection. The viral titers (C) and expression of aMPV/C N and NCL (D) were assessed using western blot, qRT−PCR, and TCID50 assay. (E) After treatment of cells with AS1411 or CRO for 72 h, western blot was utilized to detect the expression of NCL in Vero cells. (F) Vero cells were treated with NCL−siRNA for 24 h and then infected aMPV/C in the presence of AS1411 or CRO. Western blot analysis was used to detect the expression of aMPV/C N and NCL. (G) Vero cells were transfected with pCMV−HA−F for 36 h in the presence of AS1411 or CRO. The cell lysates were extracted, and an IP assay was performed using HA mAb, followed by immunoblotting with mAbs against HA and NCL. β−actin or GAPDH was used as internal control. *** p < 0.05 were considered…

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  Figure 6. The central domain of NCL plays a critical role in viral replication. (A) The structural representation for constructing full−length GFP−tag NCL and NTD, RBD, CTD, NTD−deficiency, and CTD−deficiency domains of NCL. (B) After 24 h of transfection with GFP−tag NTD, RBD, and CTD domains of NCL in Vero cells, the GFP−signal was tested by confocal microscopy. Scale bar, 100 μm (C–E) Vero cells transfected with GFP−NCL with the indicated truncations were infected with aMPV/C for 48 h for analysis of aMPV/C N expression in western blotting. (F) pCMV−HA−F and GFP−NCL with the indicated truncations were co−transfected into HEK293 cells, a Co−IP assay was performed on cell lysates using GFP mAb, followed by immunoblotting with mAbs against HA and GFP. β−actin or GAPDH was used as an internal control.