Fig 1.
PRV recognizes both human (HU-nectin-1) and swine (SW-nectin-1) nectin-1 for cell entry.
(A) PRV infects cells expressing HU- and SW-nectin-1, but not cells expressing human HVEM (HU-HVEM). CHO-K1 cells were transfected for transient expression of HU-, SW-nectin-1 and HU-HVEM, and subsequently infected with a PRV vaccine strain. Clear cytopathic changes were observed with HU- and SW-nectin-1, which are marked with arrows. (B) HU- and SW-nectin-1 mediate the gD-dependent cell fusion. A cell-based fusion assay was set up with CHO-K1 cells as previously applied in the HSV studies[21, 22]. CHO-K1 cells expressing PRV gD/gB/gH/gL and T7 luciferase were mixed and incubated with those expressing T7 polymerase in combination with HU-/SW-nectin-1 or HU-HVEM. The resultant luciferase activity was calculated and quantitatively compared with that observed for SW-nectin-1 (determined as 100% cell fusion). The results of three independent experiments were histogrammed as the means ± standard deviations (SD).
Fig 2.
An intimate binding between PRV gD and HU-/SW-nectin-1.
(A) A schematic picture of PRV gD. The boundaries of the domain elements, including the signal peptide (SP), the ectodomain, the transmembrane domain (TM), and the cytoplasmic domain (CP), were determined by bioinformatic predictions using the SignalP4.1 and TMHMM web-server. For recombinant expression of PRV gD in insect cells, the protein was truncated (aa 1–337 for gD337 and aa 1–284 for gD284), engineered with a GP67 signal peptide for secretion, and added with a C-terminal 6His tag for purification. (B) Representative size-exclusion chromatographs of gD337 and gD284. The recombinant PRV gD proteins were purified from the supernatants of the baculovirus-infected insect cells, analyzed on a Hiload 16/60 Superdex 200 column (GE), and then examined by electrophoresis by SDS-PAGE. The resultant separation profiles are shown. (C) An SPR assay characterizing the PRV-gD/nectin-1 binding kinetics. Gradient concentrations of gD337 or gD284 were flow through SW- and HU-nectin-1 immobilized on the chip surface. The real-time binding profiles are recorded and shown.
Table 1.
Data collection and refinement statistics.
Fig 3.
Structure of the unbound PRV gD.
(A) Cartoon representation of the overall structure. The PRV gD structure is composed of an IgV-like core and the surface-exposed N- and C-terminal extensions, which are colored green, orange, and magenta, respectively. The secondary elements referred to in the text are labeled. The three disulfide bonds and the N- and C-termini are marked. (B) Superimposition of the PRV (green), HSV-1 (orange), and HSV-2 (magenta) gD structures. The shaded circles and the arrows mark the variant secondary structure elements and the significant conformational differences between PRV gD and its HSV homologs. The right panel is rotated along a vertical axis for about 135 degrees to highlight the large orientation variance observed for the terminal loops. (C) A structure-based sequence alignment for PRV gD and its HSV homologs. The spiral lines and the horizontal arrows indicate α-helices and β-strands, respectively. The conserved cysteine residues that form disulphide bonds in both the PRV and the HSV gD structures are highlighted and marked numerically. For clarity, sequences of the signal peptide sequence, the C-terminal membrane-proximal loop, and the transmembrane and cytoplasmic domains were not included for the comparison. The residue numberings are based on the gD sequences of the mature proteins.
Fig 4.
Structure of the PRV-gD/SW-nectin-1 complex.
(A, B) Cartoon representation of the overall structure. The gD molecule is colored as in Fig 3A, and the membrane-distal IgV domain of SW-nectin-1 is shown in cyan. Those elements referred to in the text, including the secondary structure elements of SW-nectin-1 IgV and the interface elements in PRV gD, are labeled. The free PRV gD structure (in gray) was also aligned to the complex structure in (B) to highlight the reorientation of the gD N-terminal loop upon receptor binding. (A) The complex structure of PRV gD bound to SW-nectin-1. (B) The same complex structure that is shown after horizontal rotation of about 180 degrees. (C) Comparison of the PRV-gD/SW-nectin-1 (PRV gD in green and SW-nectin-1 is cyan) complex structure with previously reported HSV-1-gD/HU-nectin-1 (HSV-1 gD in yellow and HU-nectin-1 in orange) and HSV-2-gD/HU-nectin-1 (HSV-2 gD in magenta and HU-nectin-1 in gray) complex structures. The CC' loop of variant conformations in nectin-1, the 3.5 Å shift between the bound PRV and HSV gDs, and the unique α2' helix in PRV gD bulged towards the CC' loop of nectin-1 are highlighted and labeled. For clarity, the view of the structure in panel (C) is clockwise rotated along the vertical axis for about 90 degrees relative to that in panel (B).
Fig 5.
The atomic interaction details at the PRV-gD/SW-nectin-1 interface.
(A) An overview of the binding interface. The nectin-1 receptor is shown in surface and the gD ligand is presented as ribbons. The interface components in PRV gD, including the N-loop, the α1' helix in the IgV-like core, and the α2', α3', α3 helices and the α3'/α3 intervening loop in the C-terminal extension, are highlighted and marked with patch numbers 1–3. The amino acid interaction details for each of the three patches were delineated in panels (B), (C), and (D), respectively. (B) The interaction of the gD N-loop with nectin-1. (C) The interaction of the gD α1' and α2' helices with nectin-1. (D) The interaction of the gD α3', α3 helices and their intervening loop with nectin-1. The residues referred to in the text are shown and labeled. Dark dashed lines indicate strong H-bonds (<3.0 Å), while orange ones represent weak H-bonds (3.0–3.5 Å). (E) Amino acid sequence alignment between HU- and SW-nectin-1 highlighting their IgV domains that are recognized by PRV gD. The residues interfacing with gD are marked with black stars. For clarity, only those that contribute >2 inter-molecule Van der Waals contacts were selected. A full list of the interface residues were summarized in Table 2.
Table 2.
Comparison of the nectin-1 residues contacting PRV gD with those interfacing with HSV-1 and -2 gDsa.
Fig 6.
Mutation of the key interface residues in nectin-1 undermines the interaction with PRV gD.
(A) SPR tests of the binding between nectin-1 mutants and PRV gD337. The kinetic profiles are recorded and shown. (B) Decreased cell fusion with the mutated nectin-1 receptors. CHO-K1 cells expressing PRV gD/gB/gH/gL and T7 luciferase were mixed and incubated with those expressing T7 polymerase in combination with wild type or mutant HU-nectin-1. The histogram shows the efficiencies of cell fusion with the indicated nectin-1 mutants in comparison to that with the wild type receptor. The results are expressed as means ± SD from three independent experiments.