Structure of the aminoterminal domain of the birnaviral multifunctional VP3 protein and its unexplored critical role

Figures

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  Fig. 1.. Characterization of IBDV VP3 constructs. A) Schematic representation of VP3 (residues 1 to 257), the domain definition (D1–D3), and the recombinant protein constructs expressed in E. coli. B) Coomassie-stained SDS–PAGE gels loaded with the recombinant purified proteins used in this study. C) SEC-MALS profiles of VP3 constructs, in buffer 50 mM Tris pH 8, 500 mM NaCl, 5 mM DTT. The lines intercepting each peak correspond to the measured molecular masses shown in the right axis (VP31–221: 52 ± 0.3 kDa; VP31–81: 20 ± 0.2 kDa; VP389–221: 16.1 ± 0.03 kDa). The theoretical MWs for each construct monomer are 24.8, 13.9, and 11.0 kDa, respectively. D) Plot of the first derivative of fluorescence signal obtained in DSF experiments to measure the VP3 constructs stability at increasing temperatures (VP31–81, green; VP389–221, red; VP31–221, violet). Dashed line is indicating 50 °C. E) SDS–PAGE of the different VP3 constructs crosslinked with BS3. F) Bar plot of the quantification by densitometric analysis of SDS–PAGE gel showing that BS3 covalently stabilizes dimers and higher MW species of constructs VP31–221 (93.3 ± 3.2%), VP31–81 (96.9 ± 1.7%), and VP389–221 (43.5 ± 15.4%).

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  Fig. 2.. Structure of the IBDV VP3 aminoterminal domain. A) Ribbon representation of the monomeric structure of full-length VP3 (residues 1–257) predicted by AF3. The red dot highlights the aminoterminal end. B) As in A, but for the prediction of dimeric VP3. The aminoterminal domain (D1) is highlighted with the dashed lines box. C) Ribbon diagram of the structure of the VP3 D1 domain. Chains A and B of the D1 dimer are represented, with the secondary structural elements (α1 to α4) explicitly labeled and the aminoterminal ends depicted as red dots. D) Electrostatic potential of the protein surface showing from red to blue the negative to positive potential respectively. E) D1 monomer–monomer interactions, the molecular lipophilicity potential of protein surface is represented, from dark cyan to dark goldenrod colors, showing the hydrophilic to hydrophobic residues, respectively. Amino acids involved in monomer–monomer contacts are labeled and lateral chains showed with stick representation.

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  Fig. 3.. SAXS analyses of the IBDV VP3 constructs. A) Scattering intensity as a function of the momentum transfer Q measured for VP31–81 (green), VP389–221 (red), and VP31–221 (purple). The intensities are displaced along the y-axis for better inspection. Black lines correspond to the structural fitting of the data using different approaches (see main text). In the case of VP31–221, comparison with the simulation (dashed line) is shown together with the fitting after the Bayesian inference reweighting (solid line). The point-by-point residuals of each fit are shown at the bottom of the panel with the same color code. The inset displays the Guinier region for each one of the experimental profiles. B) Radius of gyration (Rg) distribution of the EOM-derived subensembles (green) of VP31–81 compared with that of the initial pool of conformations computed with RanCh (black). C) Subensemble of 20 conformations of VP31–81 derived from the EOM fit. The two chains of the dimer are colored in two different colors. D) Rg distribution of the conformations from the trajectory computed for VP389–221 using all-atom MD simulations, which describe the data without the need for further optimization. The…

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  Fig. 4.. The D1 of IBDV VP3 is essential for the virus life cycle. A) Schematic representation of the RG system encoding each of the IBDV genomic segments in plasmids (pCAGEN-SegA, pCAGEN-SegB) and the experimental set-up. Both plasmids were transfected in QM7 cells and virus production was analyzed by monolayer visualization and staining. Genomic Segment A with a deletion of the N-terminal domain (pCAGEN SegA VP389–257) was co-transfected with pCDNA3-VP3 encoding the full-length WT protein “Created in BioRender. Gimenez, M. (2024) BioRender.com/i89z961”. B) Western blot analysis of the transfected cells with the RG plasmids. C) Purified IBDV virus produced with the RG system. D) Bright field micrographs of QM7 monolayers 96 h posttransfection. E) Titration of extracellular viruses of cells transfected with the RG systems using the plaque assay, as described in Materials and methods. Images are representative of three independent trials. F) Bar graph shows the mean viral titers of three independent trials expressed in PFU/mL. Error bars show SD. *P < 0.05.

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  Fig. 5.. The IBDV VP3 D1 domain is crucial for capsid morphogenesis and for VP3 targeting of EEs. A) Western blot analysis of VP3 in input samples and purification (left panel), fractions of purification gradient (fractions 1 to 12 from top to bottom in gradient) for WT (central panel), and VP389–257 (right panel) polyprotein. B) Left panel, TEM of a QM7 cell transfected with the RG system WT. The inset depicts a typical cytoplasmatic paracrystalline array of IBDV viruses. Scale bar represents 2.5 µm. Right panel, electron microscopy images of purified WT VLPs expressed with recombinant vaccinia virus (fraction 8 of the gradient showed in A). Scale bar represents 200 nm. C) Left panel, TEM of QM7 cells transfected with the RG system VP389-257. Right panel, electron microscopy images of sample corresponding to fraction 5 of the gradient shown in A. D) Immunofluorescence labeling showing the subcellular distribution of IBDV VP3 and VP2 in QM7 cells transfected with RG WT or VP389–257 plasmids (segments A). Scale bars represent 5 µm E) Immunofluorescence labeling showing the subcellular distribution of the above indicated VP3 constructs in QM7 cells co-expressing EGFP-Rab5 WT. Scale bars…

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  Fig. 6.. IBDV VP3 contacts and capsid assembly model. A) Full-length VP3 dimeric model generated by AlphaFold (28) combined with crystal structures (PDB ID: 2R18). Protein flexibility is depicted in semitransparent. Regions involved in interdomain contacts are indicated with a dashed arrow and flexible regions with the symbol (right panel). B) Model for VP3 multimerization mediated by D1 and by the contacts between D2 and D3 of neighbor dimeric molecules, mimicking the antiparallel arrangement observed in D2–D3 crystals (24 and P212121 structure of this work), forming highly flexible elongated oligomers. The dashed box in the right panel is a magnification of the selected region of the model. The bottom panel shows a schematic representation of the model. C) Contribution of VP3 dimerization to the IBDV capsid assembly. Model of the dimeric VP3 interacting with VP2 trimers. While one or two VP3 C-terminal tails of one VP3 dimer are interacting with one or two VP2 trimers, the N-terminal domains of VP3 are maintaining its dimeric state. At the same time, some C-terminal tails or the middle domains (D2–D3) of a neighbor VP2–VP3 complex can establish contacts with the first complex, pulling…