Fig 1.
Classification scheme of particles identified in cryo-EM micrographs and validation of subnanometer structures of either 93k (A to E) or SG2 (F to J) Fab fragments in complex with purified VZV gB. B to J–Single particle cryo-EM micrographs of 93k (B and C) or SG2 (G and H) Fab fragments in complex with VZV gB in vitreous ice on lacey carbon (B and G) or Quantifoil (C and H) grids captured with a 200kV F20 (FEI). Representative 2D class averages are shown. Fourier shell correlation plot (D and I) and Euler angle distribution (E and J) of VZV gB-93k or gB-SG2 complex particles included in the 3D reconstruction.
Fig 2.
Subnanometer resolution cryo-EM structures of Fab fragments from either 93k or SG2 in complex with native, full-length VZV gB purified from VZV infected MeWo cells.
A to C–Subnanometer resolution cryo-EM maps of gB-93k (93k –blue; 7.3Å) and gB-SG2 (SG2 –green; 9.0Å) complexes focused at the gB-Fab fragment interface (A and B), viewed from the side and top (C) and a composite cryo-EM map of the 93k and SG2 Fabs bound to native gB showing the difference in binding angles of the two Fabs. The Fab binding angles were calculated from the central axis of gB (vertical line) and the central axes of the 93k and SG2 Fabs. The gB ectodomain is shown in grey and the CTD show in red; the CTD is depicted at a different threshold to the ectodomain due to the lower resolution for this part of the cryo-EM map. D–A linear map of VZV gB showing the location of the mAb 93k major binding sites (β23 and β30) and the predicted mAb SG2 binding site (β25 and β26) in gB DIV. The gB furin cleavage site is shown. VZV gB domains are colored as follows, DI (cyan), DII (green), DIII (yellow), DIV (orange), DV (red) and linker regions (hot pink).
Fig 3.
Anti-VZV gB monoclonal antibody interactions with gB DIV predicted from 93k-Fab and SG2-Fab subnanometer cryo-EM structures.
A to C–Cryo-EM maps of Fab fragments (93k at 2.8Å [33]–light blue; 93k at 7.3 Å–blue; SG2 at 9.0Å –green) bound to VZV gB DIV (orange). Interactions between Fab fragments from either 93k or SG2 with gB DIV domain (orange). The 93k and SG2 interactions are mapped on the gB DIV domain (orange ribbon), directly from segmented density of the Fab in maps of gB-93k 2.8Å (A–cyan), gB-93k 7.3Å (B–blue), and gB-SG2 9.0Å (C–green). Residues within 6Å of the segmented density in each respective case are colored on the ribbon and highlighted in the corresponding amino acid sequence below the cryo-EM and ribbon structures. Blue indicates residues that are predicted to interact with 93k, green indicates residues that are only predicted to interact with the SG2 Fab and not 93k (both 2.8Å and 7.3Å). Cryo-EM map thresholds: A– 0.03; B– 0.422; C– 2.52.
Fig 4.
Single particle cryo-EM of native, full-length VZV gB.
A–Classification scheme of VZV gB particles identified in Cryo-EM micrographs purified VZV gB frozen on lacey carbon grids. B–Native PAGE of purified VZV gB and western blots with VZV gB specific rabbit IgG 746–868. Molecular weights of the protein standards are given to the left of the gel (kDa). C–Single particle cryo-EM micrograph of a lacey carbon grid with purified VZV gB post size exclusion chromatography with four representative 2D class averages. D–Fourier shell correlation plot and Euler angle distribution of VZV gB particles included in the 3D reconstruction. E–Distribution of resolution (Å) of the VZV gB cryo-EM map calculated by ResMap.
Fig 5.
Near atomic resolution structures of VZV gB native, full-length VZV gB purified from VZV infected MeWo cells and the ectodomain transiently expressed in HEK293 GnTl- cells.
A–A diagram of the linear structure for VZV gB expressed by the pOka-gB-TEVV5 virus. The signal sequence is depicted by the crossed white box. Colored regions between residues 115–736 represent those that were resolved in the cryo-EM structure; DI (cyan), DII (green), DIII (yellow), DIV (orange), DV (red) and linker regions (hot pink). Disulphide bonds are represnted by the connecting lines. The colored regions beyond residue 931 represent the tag used for purification of gB from infected cells; TEV cleavage site (lime green) and V5 tag (blue). B–The cryo-EM map of native, full-length VZV gB constrained to C3 symmetry (3.9Å). The ectodomain is based on a focussed refinement map with each protomer of the gB ectodomain highlighted in different colors (blue, white and green) and the CTD represented at a lower threshold (pink). C–A ribbon diagram for the gB ectodomain structure is colored as for the diagram in A. D–Segmentaion and MapQ analysis of the VZV gB cryo-EM map based on a single protomer of the gB ectodomain. The scale, red to green (0 to 1), represents the goodness of fit of the cryo-EM map with the structure using MapQ. E–The linear structure of the truncated VZV gB used for X-ray crystallography. The signal sequence is depicted by the crossed white box. Colored regions between residues 115–736 represent those that were resolved in the gB crystal structure from transiently transfected cells; DI (cyan), DII (green), DIII (yellow), DIV (orange), DV (red) and linker regions (hot pink). The location of the residues that were substituted in the fusion loop (W180G and Y186G) and the furin cleavage site (481GSGG484) of the gB ectodomain expression construct used for X-ray crytsallography are indicated. The white regions represent portions of gB that were not resolved in the crystal structure. The grey shaded box indicates the truncation. The clubs represent glycosylation sites in the X-ray crystallography data (black) or by orbitrap mass spectrometry (red). F to H–The X-ray crystal structure of VZV gB ectodomain at 2.4Å. Ribbon diagrams of the gB trimer (F), monomer (G) and the monomer superimposed with a portion of a segmentation of the 3.9 Å cryo-EM map.
Fig 6.
Peptide coverage from native, full-length VZV gB digested with trypsin and detected by Orbitrap mass spectrometry.
The pOka-gB[ORF31] line shows the domain colors based on the X-ray crystal structure. MassSpecCoverage shades the gB sequence (green, black text) to show the composite peptide coverage generated by trypsin digest and detected by Orbitrap mass spectrometry. TrypsinCleavageSites shades the K/R cleavage sites (green, red text) to show the location of potential trypsin cleavage sites in VZV gB. The NXS/T line shows the location of predicted N-linked glycosylation sites in the gB ectodomain.
Fig 7.
Structure based amino acid alignments of herpesvirus gB orthologues.
The amino acid sequences given are the partial ones used to express the gB homologs for crystallography studies and are numbered according to the position in the full-length proteins. HSV (HHV1[2GUM] [5]), PRV ([6ESC] [34]), HCMV (HHV5[5CXF] [3]) and EBV (HHV4[3FVC] [2]) compared to VZV (HHV3[6VLK]). The VZV gB domains are colored as per the crystallography DI (cyan), DII (green), DIII (yellow), DIV (orange), DV (red) and linker regions (hot pink). Residues colored with white text and a blue background in the VZV gB amino acid sequence were subsituted for glycine (G) in the transient expression constructs used for X-ray crystallography. Residues colored with white text and a black background were not resolved in the X-ray crystallography data for each of the herpesvirus gB orthologues. The consensus sequence below the alignment depicts conserved (upper case) and partially conserved (lower case) residues in the gB amino acids sequences. The sequences are numbered according to the complete gB for each herpesvirus.
Fig 8.
The N-termini of PRV and low pH HSV gB have an ordered structured compared to those of VZV and neutral pH HSV.
The gB DIV structures for VZV (PDB), PRV (6ESC[34]), HSV (2GUM[5]) and low pH HSV (3NWF[87]) gB are represented in ribbon format. Residues in the gB structure for the polypeptide chain are indicated. The ordered N-terminal regions of PRV and low pH HSV gB are highlighted by the dotted boxes.
Fig 9.
Homology model of the VZV gB N-terminus.
Residues 77–114 of VZV gB were subject to homology modelling using RaptorX [88]. The predicted α-helix is colored yellow.
Fig 10.
The gB N-terminus is critical for cell-cell fusion and VZV replication.
A–Near atomic structure of the gB N-terminus within the footprint of mAb 93k [33]. The orientation of the complete gB-93k map is shown in the small box in the top left-hand corner colored as for Fig 1E of [33]. A portion of the cryo-EM map for gB domain IV chain A (Orange) and the bound 93k Fab (Blue) are shown. The red box on the right is a magnified view of the location of the N-terminus with the amino acid side chains (grey) shown for VZV gB and 93k VH and VL chains. Amino acid side chains of the gB N-terminus that interact with 93k VL chain are colored green and interacting atoms connected with a dotted magenta line. The structure for the gB N-terminus is not modelled beyond T115 due to the lack of structure identified for both the cryo-EM map and X-ray crystallography data. B–Quantification of total and cell surface gB DIV mutants produced by transfected CHOs and their capacity for cell-cell fusion measured by the SRFA. All values are normalized as a percentage to WT gB. Bar charts represent n = 4 samples for total and cell surface gB detected using mAbs SG2 and 93k, and at least n = 24 samples for fusion examined over 2 independent experiments. Error bars represent ±SEM. C–Immunofluorescence of MeWo cells at 72 hours post transfection with pOka-BACs with gB mutations. Melanoma cells were transfected with pOka-TK-GFP BACs carrying alanine substitution at K109A, K109R, S110A, Q111A, D112A and 109AAAA112. The (+) or (-) indicate whether virus was recovered or not, respectively, from the transfections. Immunofluorescence staining was performed for IE62 as a marker for early infection because the TK-GFP is a late protein product during VZV replication. Scale bar (white) 100μm. D–Immunohistochemistry staining of plaques and their sizes for the pOka-TK-GFP gB, pOka-TK-GFP gB-STEVV5 and N-terminal mutants K109A, K109R, S110A, Q111A, D112A and 109AAAA112. Scale bar (black) 1mm. Bar charts represent n = 40 plaques measured over two independent experiments. All values were normalized to WT VZV. E–Immunoprecipitation (IP) of the VZV gB N-terminal mutants from transfected CHO cells using anti-gB mAbs SG2 and 93k, and western blot with anti-gB Ab 746–868. The gH lane is a control where CHO cells were transfected with gH-WT. F–Reducing SDS PAGE and western blot of gB co-immunoprecipitated with gH-V5 from CHO cells transfected with the N-terminal mutants, gH-V5 and gL. The first gB-WT lane is a control lane using gH-WT. Western blots were performed using mAb to V5 (Top; WB-V5), anti-gB Ab 746–868 (middle; WB-gB) and a mAb 93k (bottom; WB - 93k). E and F–Numbers to the right of the blots are molecular weight standards (kDa). The red arrows and corresponding numbers indicate molecular weights for gH (118, 100 and 75 kDa; V5 tagging of gH enables the detection of different maturation states of the glycoprotein [89]) and gB (130kDa; only the mature uncleaved form of VZV gB interacts with VZV gH).
Fig 11.
The neutralizing epitope of mAb 93k is accessible on the prefusion form of VZV gB.
A–A homology model of VZV gB in a prefusion conformation based on the 9.0Å cryo-EM structure of HSV gB [37]. The left-hand panel shows a single VZV gB protomer and the right-hand panel is the complete gB trimer in the prefusion conformation. The linear structure of VZV gB below the two panels depicts the colors used for the homology model; the signal sequence (crossed white box), DI (cyan), DII (green), DIII (yellow), DIV (orange), DV (red), linker region (hot pink) and CTD (grey box). B and C–Snapshots taken from S4 Movie. B–The prefusion conformation of VZV gB modelled in conext with a lipid bilayer composed of lipids SM (3.1%), PC (51.2%), PI (11.3%), PS (4.6%), PE (29.8%) found in herpesvirus virions [85]. C–The mAb 93k (blue) and SG2 (green) Fabs bound to the prefusion conformation of VZV gB. Domian IV (orange) of VZV gB is represented in surface. The presence of the lipid bilayer does not prevent accesibility of the 93k epitope but will prevent SG2 from binding to gB in the prefusion conformation. The left-hand panel shows a rotated (90°) and zoomed in region of gB DIV and the footpring of the 93k and SG2 Fabs as determined in Fig 3.