Biophysical characterization of VP3 binding to PI3P.

(A) Immunoblots of the top (T) and bottom (B) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-2xFYVE protein specifically binds to liposome PI3P(+). Results are representative of three independent experiments. The bar plot represents the intensity of T/B bands for each liposome preparation. Significant differences (** P <0.01) as determined by one-way ANOVA with Tukey’s HSD test.

(B) Immunoblots of the three top (T1, T2 and T3) and bottom (B) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 FL protein specifically binds to liposome PI3P(+). Results are representative of three independent experiments. The bar plot represents the intensity of (T1+T2+T3)/B bands for each liposome preparation. Significant differences (* P <0.05) as determined by one-way ANOVA with Tukey’s HSD test.

(C) Cryo-electron microscopy images of cryo-fixated liposomes PI3P(+) control (without protein), or incubated with His-2xFYVE- or His-VP3 FL-Ni-NTA gold particles showing gold particles decorating the membrane of the liposomes when His-2xFYVE or His-VP3 FL were present. The bar represents 50 nm.

(D) Binding of His-Streptavidin (negative control), His-2xFYVE (positive control) or His-VP3 FL to three different concentrations of liposomes PI3P(-) or PI3P(+). Association and dissociation sensorgrams measured by bio-layer interferometry (BLI), showing the specific interaction of His-2xFYVE and His-VP3 FL with liposomes PI3P(+) in a dose-dependent manner, as indicated.

(E) Cartoon representation of AlphaFold prediction of VP3 FL. Red region corresponds to the “core” region of the protein present in the experimental X-ray crystallographic model obtained by Casañas et al., 2008 [PDB: 2R18 (Casañas et al., 2008)]. Regions not present within the PDB are colored in violet and blue representing the Nt and the Ct of VP3, respectively. pLDTT values lower than 50 are a strong predictor of disorder.

(F) Binding of His-VP3 DCt to three different concentrations of liposomes PI3P(-) or PI3P(+). Sensorgrams measured by BLI, showing the absence of binding to either liposomes when the VP3 lacks the Ct region [blue in (E)].

(G) Immunoblots of the three top (T1, T2 and T3) and bottom (B) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay of His-VP3 FL protein (positive control, left panel) or His-VP3 DCt (right panel) showing the lack of VP3 DCt binding to both liposomes. Results are representative of three independent experiments. The bar plot represents the intensity of (T1+T2+T3)/B bands for each liposome preparation. Significant differences (* P <0.05; ns P >0.05) as determined by one-way ANOVA with Tukey’s HSD test.

VP3 P2 involvement in the association of VP3 with the EE membrane.

(A) The cartoon representation of the VP3 dimer [PDB 2R18 (Casañas et al., 2008)] shows each protomer in different shades of grey. The blue balls depict the residues defining the P2 region.

(B) The close-up of P2 region showing residues K157, R159, H198 and R200.

(C) Electrostatic potential mapped on the surface of the structure of a VP3 dimer in the same orientations as in (A) structures. The close-up shows the impact of P2 residue mutations on the electrostatic potential of the binding site. P2 WT corresponds to the “UniProt code”. The color-coded electrostatic surface potential of VP3 was drawn using PyMol (blue positive, red negative).

(D) QM7 cells transfected with pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four point mutants (K157D, R159D, H198D and R200D) and immunostained with anti-VP3 showing the distribution of each protein (upper panel). Images were captured using a Confocal Laser Scanning Microscopy and then the percentage of cells with punctated fluorescent signal were determined for each protein (lower panel). The red signal shows the VP3 distribution and the blue one shows the nuclei, which were Hoestch-stained. The data were normalized to the P2 WT protein. The box plot represents the percentage of cells with punctuated distribution of VP3. Significant differences (*** P <0.001; ns P >0.05.) as determined by one-way ANOVA with Tukey’s HSD test.

(E) QM7 cells co-transfected with pEGFP-Rab5 and pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four point mutants (K157D, R159D, H198D and R200D) and immunostained with anti-VP3 showing the distribution of each protein. Representative images, captured using a Confocal Laser Scanning Microscopy are shown where green signal represents Rab5 distribution and the red signal that of VP3. Nuclei were Hoestch-stained and are blue. White bar-scales represent 20 nm. VP3 P2 (all reversed) and R200D depict a cytosolic distribution of the proteins. Quantification of the co-localization of the different VP3 proteins and EGFP-Rab5 is shown in SI Appendix, Fig. S3.

VP3 P2 involvement in the association between VP3 and EE PI3P.

(A) QM7 cells co-transfected with the PI3P biosensor pEGFP-2xFYVE and pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four point mutants (K157D, R159D, H198D and R200D) and immunostained with anti-VP3 showing the distribution of each protein. Representative images, captured using a Confocal Laser Scanning Microscopy are shown where green signal represents FYVE distribution and the red signal that of VP3. Nuclei were Hoestch-stained and are blue (upper panel). White bar-scales represent 20 nm. VP3 P2 (all reversed) and R200D depict a cytosolic distribution of the proteins with a significant lower co-localization coefficient. The dot plot in the lower panel depicts the co-localization coefficient for each protein determined as explained in the Materials and Methods section. Significant differences (ns P >0.05) as determined by one-way ANOVA with Tukey’s HSD test.

(B) QM7 cells were transfected with the pcDNA VP3 FL (P2 WT), P2 (all reversed), or the four point mutants (K157D, R159D, H198D and R200D). The cells were fixed and then the GST-2xFYVE purified peptide and anti-VP3 antibodies were used to recognize endogenous PI3P and VP3, respectively. Additionally anti-EEA1 antibodies were used to stain the endosomes. GST-2xFYVE was labelled with a fluorescent anti-GST antibody (blue signal), anti-VP3 and anti-EEA1 antibodies with fluorescent secondary antibodies (cyan and green signals, respectively), and Hoestch-stained nuclei in orange. White bar-scales represent 10 nm. VP3 P2 (all reversed) and R200D depict a cytosolic distribution of the proteins with a significant lower co-localization coefficient. The dot plot in the lower panel depicts the co-localization coefficient for each protein determined as explained in the Materials and Methods section. Significant differences (*** P < 0.001; ns P >0.05) as determined by one-way ANOVA with Tukey’s HSD test.

Biophysical characterization of VP3 FL R200D binding to PI3P.

(A, left panel). Immunoblots of the top (T) and bottom (B) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-2xFYVE protein specifically binds to liposome PI3P(+). The bar plot represents the intensity of T/B bands for each liposome preparation. Significant differences (ns P >0.05) as determined by one-way ANOVA with Tukey’s HSD test.

(A, rigth panel). Immunoblots of the three top (T1, T2 and T3) and bottom. (B) fractions from a liposome PI3P(-) or PI3P(+) OptiprepTM co-floatation assay indicating that His-VP3 FL R200D does not bind to liposome PI3P(-) nor PI3P(+). The bar plot represents the intensity of (T1+T2+T3)/B bands for each liposome preparation. Significant differences (ns P >0.05) as determined by one-way ANOVA with Tukey’s HSD test.

(B) Far-UV CD spectra of His-VP3 FL (red line) or His-VP3 FL R200D (green line). Spectral acquisitions at 50 nm/min with 0.1 nm steps at 1 s integration time, with a bandwidth of 1 nm were performed 4 times for the samples as well as for the buffer. The measurements were carried out with constant nitrogen gas flux of 10 ml/min. Acquisitions were averaged and buffer baseline was subtracted with Spectra Manager (JASCO). No smoothing was applied. CDtoolX was used to zero between 255-260 nm and to calibrate the signal amplitude from the fresh CSA signal (Miles and Wallace, 2018). Data are presented as delta epsilon (Δε) per residue (L.mol-1.cm-1.residue1) calculated using the molar concentration of protein and number of residues.

(C) QM7 cells were grown in M24 multi-well plate for 12 h to approximately 90-95% confluency and then 800 ng of plasmids were transfected [(SegA + SegB) or (SegA.R200D + SegB)] in triplicate. At 8 h post-transfection (p.t.) the supernatants were discarded, and the monolayers were recovered for further plating on M6 multi-well plates containing non transfected QM7 cells. Avicel RC-591 (FMC Biopolymer) was added to the M6 multi-well plates. 72 h p.i., the monolayers were fixated and stained with Coomassie R250 for revealing the foci forming units.

(D) Partial view of amino acid alignment of the VP3 protein for nine reference members of Birnaviridae family. Multiple sequence alignment was performed with Clustal OMEGA (v1.2.4) (Sievers et al., 2011) implemented at EMBL’s European Bioinformatics Institute (“EMBL’s European Bioinformatics Institute,” n.d.) (The complete alignment is shown in SI Appendix, Fig. S4A). Alignment visualization was done with the R ggmsa package (Zhou et al., 2022) in with assistance from the RStudio software (RStudio Team, 2020). Amino acids are colored according to their side-chain chemistry. Protein sequence logos annotation is displayed on top of the amino acid alignment. For facilitating the view IBDV VP3 142-210 portion is shown. The black arrow indicates the K157, R159, H198 and R200.

Adsorption of VP3 constructs to PI3P(+) model membranes.

(A-C). Adsorption free-energy profiles for VP3 Ct, VP3 Δ81 and VP3 FL at 50 and 150 mM of NaCl.

(D) Adsorption free energy (ΔGad), computed from the minimum of PMF(z), versus concentration of NaCl. In all cases, the solution pH is 8, and the concentration of protein is 1 µM. The grey areas represent the volume excluded by half the membrane (cis hemilayer, z>0). The membrane surface contains 5% titratable groups, representing PI3P. Each group has three acidic moieties, one with a pKa of 2.5 and the others with 6.5. At 150 mM NaCl and pH 8, more than 90% of the acidic groups are deprotonated (SI Appendix, Fig. S5).

VP3 approaching a lipid bilayer, and distortion of the membrane.

(A-D) Temporal sequence of configurations illustrating how VP3 D81 approaches the negatively charged membrane during a 500 ns MD simulation. The membrane contains DOPE, DOPC and PI3P in 64:31:5 molar ratio. The beads making the Ct fragment are colored blue.

(E) Magnification of the protein configuration bound to the membrane, with P2 residues represented in cyan balls.

(F) Radial distribution function, g(r), between the center of mass of VP3 D81 and the center of PI3P molecules in the cis hemilayer. g(r) greater than 1 implies local enhancement of PI3P concentration. The inset shows an upper view of VP3 D81 bound to the membrane, with the area within r = 4 nm colored in light gray.

(G) Cryo-electron microscopy images of cryo-fixated liposomes PI3P(+) control (without protein, left panel), or incubated with His-VP3 FL showing the small pinches or localized thinnings in the bilayer of the liposomes when His-VP3 FL were present (middle panel). The bar represents 50 nm. The right panel represents an enlarged image of the red square on the middle panel. White arrows point the small pinches or localized thinnings in the bilayer of the liposomes when His-VP3 FL were present.