Structural Insights Into the Nuclear Import of Marek’s Disease Virus Large Tegument Protein

Figures

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  FIGURE 1. Interaction of GaAHV2 UL36 NLS with various IMPs. (A) EMSA demonstrating the binding of the GaAHV2 UL36 NLS peptide to different IMPα isoforms. The NLS peptides were labelled with FITC and an Ahx linker, allowing visualization under UV light (green fluorescence). Protein bands were detected by Coomassie blue staining (blue). The EMSA data represent results from three independent experiments. (B) FP assay assessing the binding affinity between the GaAHV2 UL36 NLS and the corresponding IMP isoforms. Values are presented as the mean ± standard error (SE) from three separate experiments. Binding constants (K D ) were determined according to the procedures outlined in the Materials and Methods section.

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  FIGURE 2. Resolved crystal structure of IMPα bound to GaAHV2 UL36 NLS. (A) The IMPα structure (grey cartoon) displays the GaAHV2 UL36 NLS peptide at the major binding site (pink sticks) and minor binding site (aquamarine sticks). (B) Schematic representation of the interactions between GaAHV UL36 NLS and IMPα, with residues at the major binding site highlighted in pink and those at the minor binding site in aquamarine. Interfacing amino acids from IMPα are indicated by grey boxes, with salt bridges denoted in bold text. Defined cargo binding pockets are labelled in white. (C) Close‐up images of GaAHV UL36 NLS amino acid side chains (pink) and IMPα interfacing residues (grey sticks) with interactions calculated by PDBePISA, showcasing binding pockets P2–P5. (D) Close‐up images of GaAHV2 UL36 NLS amino acid side chains (aquamarine sticks) and IMPα interfacing residues (grey sticks), also with interactions calculated by PDBePISA, highlighting binding pockets P1′–P4′. Molecular image generated in Pymol.

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  FIGURE 3. Binding affinities of IMPα2 with GaAHV2 UL36 NLS mutants. (A) EMSA analysis showed no detectable binding between IMPα2 and the NLS mutant peptides, except for the R472A mutant. Each peptide was labeled at the N‐terminus with FITC and an Ahx linker and visualized under UV light (green). Proteins were stained with Coomassie blue, and the overlay images display both the FITC‐tagged peptides and the Coomassie‐stained IMPα proteins. The EMSA results represent data from three independent experiments. (B) Fluorescence polarization assays quantified direct binding interactions between the NLS mutants and the indicated IMPα2 isoform. K D values are presented as mean ± standard error of the mean (SEM) from three separate experiments. Nonlinear regression analysis for K D calculation was performed using GraphPad Prism, as detailed in the Materials and Methods section.

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  FIGURE 4. Interaction analysis of IMPα1 major and minor site mutants with GaAHV2 UL36 NLS. (A) EMSA indicates a lack of binding between the GaAHV2 large tegument protein NLS and the IMPα1 mutants at both the major and minor binding sites. The GaAHV2 UL36 NLS peptides were labeled at the N‐terminus with FITC and included an Ahx linker, allowing visualization under UV light (green fluorescence). Proteins were visualized using Coomassie blue staining (red). The EMSA results are representative of three separate experiments. (B) FP assay quantifying the binding affinity of GaAHV2 UL36 NLS to the IMPα1 major and minor site mutants. Data represent the mean ± standard error of the mean (SEM) from three independent experiments. Dissociation constants (K D ) were calculated by nonlinear regression analysis using GraphPad Prism, following the methods described in the Materials and Methods section.