Antibody blocking abrogates NF-κB activation by the HSV-1-encoded gB.

<p>(A) HEK293T cells were transiently transfected with pCMV1-FLAG-hTLR2 along with the NF-κB reporter gene plasmid. After 24–36 h, cells were pre-incubated with blocking anti-TLR2 mAb or IgG2a isotype control Ab at a final concentration of 10 µg/ml for 1 h at 37°C followed by treatment with either gBs (20 µg/ml), HD-gBs (20 µg/ml), UV-HSV-1 (MOI = 5), zymosan (10 µg/ml) or anti-gB mAb or mouse IgG preincubated gBs, HD-gBs and UV-HSV-1 at a final concentration of 10 µg/ml (37°C for 1 h) for 8 h, and luciferase reporter gene activity was measured as described in <i>Materials and Methods</i>. Luciferase activity values are expressed as the ratio between firefly and <i>Renilla</i> luciferase. The results are shown as the mean ± SD of three independent experiments. (B) to (D) Validation of NF-κB activation by gBs and UV-HSV-1 through detection of IκBα degradation. HEK293T cells transfected with TLR2 expressing plasmid for 24–36 h were pre-incubated with blocking anti-TLR2 mAb and IgG2a isotype control Ab at a final concentration of 10 µg/ml for 1 h at 37°C followed by treatment with either gBs (20 µg/ml) (B), HD-gBs (20 µg/ml) (C), UV-HSV-1 (MOI = 5) (D), zymosan (10 µg/ml) or anti-gB mAb or mouse IgG preincubated gBs (B), HD-gBs (C) and UV-HSV-1 (D) at a final concentration of 10 µg/ml (37°C for 1 h) as indicated for 8 h, then cell lysates were prepared and IκBα and actin levels were determined by 10% SDS-PAGE analysis followed by IB using anti-IκBα and anti-actin Abs (right panel). Left panel, The immunoblot shown in the right panel was subjected to densitometric analysis using Quantity One 4.4 software. The intensity of the IκBα bands for each sample was normalized to the intensity of the corresponding actin band. For each lane, quantification is expressed as the ratio of IκBα to actin. IB, immunoblotting. The results are indicative of two independent experiments.</p

Mutation of the key interface residues in nectin-1 undermines the interaction with PRV gD.

<p>(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.</p

HSV-1-encoded gB coimmunoprecipitates with TLR2 in transfected and infected cells.

<p>HEK293T cells were transiently co-transfected with 5 µg of two expressing plasmids pCMV1-FLAG-hTLR2 and pcDNA3-gB (A and B), pCMV1-FLAG-hTLR4 and pcDNA3-gB (C) or first transfected with 5 µg of pCMV1-FLAG-hTLR2 for 24 h followed by infection with HSV-1 at an MOI of 5 (D). Thirty-six hours after transfection or infection, cells were lysed and IP with anti-FLAG mAb, anti-TLR2 mAb or nonspecific mouse antibody (IgG) was performed. Immunoprecipitated proteins, as well as the cell lysates, were separated by denaturing 10% SDS-PAGE and analyzed by IB with anti-FLAG mAb or anti-gB pAb R68 as indicated. IP, immunoprecipitation. IB, immunoblotting.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

HSV-1-encoded gB signals through MyD88/TRAF6, but not TRAF2.

<p>HEK293T cells were transiently transfected with pCMV1-FLAG-hTLR2 and the NF-κB luciferase reporter plasmid along with vector (pCMV-HA or pCMV-FLAG) and various concentrations of pHA-DN-MyD88 (A), pDN-TRAF6 (B) or pFLAG-DN-TRAF2 (C). After 24–36 h, cells were stimulated with gBs (20 µg/ml) or zymosan (10 µg/ml) or left untreated for 8 h, and luciferase reporter gene activity was measured as described in <i>Materials and Methods</i>. Luciferase activity values are expressed as the ratio between firefly and <i>Renilla</i> luciferase. The results are shown as the mean ± SD of three independent experiments. Statistical analysis was performed using Student’s <i>t</i> test. (A) *, <i>P = </i>0.011. (B) *, <i>P = </i>0.023.</p

TLR2 is required for gB-mediated NF-κB activation.

<p>(A) Dose-dependent activation of NF-κB by HSV-1-encoded gB. HEK293T cells were transiently transfected with pCMV1-FLAG-hTLR2 and/or pcDNA3.1-hCD14 along with NF-κB luciferase reporter plasmids. After 24–36 h, cells were stimulated with various concentrations of gBs (0, 0.2, 2, 10, 20 and 40 µg/ml) for 8 h. (B) Time-dependent activation of NF-κB by HSV-1-encoded gB. HEK293T cells were transiently transfected with pCMV1-FLAG-hTLR2 and NF-κB luciferase reporter plasmids. After 24–36 h, cells were stimulated with gBs (20 µg/ml) for 1, 2, 4, 8, 12, and 24 h or zymosan (10 µg/ml) for 8h or left untreated. (C) Dominant-negative TLR2 dose-dependently inhibits HSV-1-encoded gB-mediated activation of NF-κB. HEK293T cells were transiently transfected with pCMV1-FLAG-hTLR2 and the NF-κB luciferase reporter plasmid along with various concentrations of pCMV1-FLAG-DN-hTLR2. After 24–36 h, cells were stimulated with gBs (20 µg/ml) or zymosan (10 µg/ml) or left untreated for 8 h, and luciferase reporter gene activity was measured as described in <i>Materials and Methods</i>. Luciferase activity values are expressed as the ratio between firefly and <i>Renilla</i> luciferase. The results are shown as the mean ± SD of three independent experiments. Statistical analysis was performed using Student’s <i>t</i> test. (A) HEK293T-TLR2, *, <i>P = </i>0.030; HEK293T-TLR2/CD14, *, <i>P = </i>0.029. (B) *, <i>P = </i>0.024. (C) *, <i>P = </i>0.043.</p

Mutation of the key interface residues in nectin-1 undermines the interaction with PRV gD.

<p>(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.</p

An intimate binding between PRV gD and HU-/SW-nectin-1.

<p>(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.</p

Structure of the unbound PRV gD.

<p>(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.</p

Structure of the PRV-gD/SW-nectin-1 complex.

<p>(A, B) Cartoon representation of the overall structure. The gD molecule is colored as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006314#ppat.1006314.g003" target="_blank">Fig 3A</a>, 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).</p