Ectopic expression of HSV-1 gE, HCMV gp68 and HCMV gp34 inhibit IgG1 (trastuzumab) mediated activation of FcγRs.

<p>(<b>A</b>) SKOV-3 cells were infected for 24 h with 2 PFU/cell VACV wt and rVACV expressing gE or (<b>B</b>) rVACV expressing gp68 or gp34 before opsonized with trastuzumab at different concentrations for 30 min. After removing of unbound antibodies by repeated washing with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR-ζ activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured, means with standard deviations (error bars) are shown for 3 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

HCMV gp68 and gp34 inhibit FcγRIII activation by rituximab antibody isotypes.

<p>CD20 transfected 293T cells were infected for 16/cell of VACV wt or rVACV expressing gp68, gp34 or MULT-1 as a control. After opsonization with 1 µg/ml of each antibody isotype for 30 min. and removing of unbound antibody by washing, cells were co-cultivated with 1×10⁵ BW:FcγRIIIA-ζ reporter cells per well for 16 h before supernatants were collected and mIL-2 was determined by ELISA. Each value represents three replicates; means with standard deviations (error bars) are shown for 2 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

Soluble ectodomains of HCMV vFcγR interfere with antibody dependent NK cell degranulation.

<p>(<b>A</b>) Cytotect was coated to a plate in binding buffer (0.1 M Na₂HPO₄ pH 9.0) at a concentration of 0.5 mg/ml and incubated for 2.5 hours at 37°C. After blocking for 30 minutes and washing unbound antibodies, soluble proteins, rIL-2 pre-activated primary NK cells and α-CD107a-PECy5 antibody were added and incubated for 4 hours at 37°C. Duplicates were measured for CD107a surface expression after dead cell exclusion with DAPI staining in a FACS Canto II. Means are shown with standard deviations (error bars). Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>B</b>) To compare the amounts of soluble proteins used in (A), SDS-PAGE and anti-V5 immunobotting was performed.</p

The presence of the vFcγRs on the surface of infected cells inhibits antibody dependent NK cell degranulation.

<p>(<b>A</b>) MRC-5 fibroblasts were infected with HSV-1F wt, ΔgE and ΔgE revertant for 24 h before cells were opsonized with HSV IgG positive and negative human sera. After 30 min of incubation, unbound antibodies were washed away and NK cells at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells was measured in FACS. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a-positive cells with the immune antibody opsonization - % of CD107a-positive cells with non-immune antibody). (<b>B</b>) MRC-5 fibroblasts were infected with HCMV HB5 wt, HB5Δgp68, HB5ΔIRLΔgp34 and HB5ΔIRLΔgp68/Δgp34 for 72 h before cells were opsonized with HCMV IgG positive and negative human sera. After 30 min of incubation, unbound antibodies were washed away and NK cells at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells was measured in FACS. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a-positive cells with the immune antibody opsonization - % of CD107a-positive cells with non-immune antibody) (<b>C</b>) MRC-5 fibroblasts were infected with HCMV HB5 wt and HB5ΔIRLΔgp68/Δgp34 for 72 h before opsonized with Cytotect or human HCMV-IgG negative sera at 1∶10 and 1∶100 dilutions. After 30 min of incubation, unbound antibodies were washed away and NK cells from six different donors at an E∶T ratio of 10∶1 were added. After 4 h, CD107a surface expression on the NK cells against HCMV-infected cells was measured. Percentage of IgG-specific degranulation CD107a-positive cells = (% of CD107a positive cells with the immune antibody opsonization - % of CD107a positive cells with non-immune antibody). Single measurements of CD107a cells are shown. One of three (A, B) or two (C) representative experiments is shown.</p

Soluble ectodomains of HCMV vFcγRs interfere with FcγR activation.

<p>(<b>A</b>) SKOV-3 cells were opsonized with 100 ng/ml trastuzumab for 30 minutes and washed three times with D-MEM 10% FCS (vol/vol) before soluble proteins were added in graded concentrations concomitantly with BW:FcγRIIIA-ζ transfectants. mIL-2 was determined in supernatants (which were harvested after 16 h of co-cultivation of responder cells with target cells) by ELISA. (<b>B</b>) As in (A) but SKOV-3 cells were opsonized with 500 µg/ml trastuzumab for 30 minutes and washed three times with D-MEM 10% FCS (vol/vol) before soluble proteins were added in graded concentrations concomitantly with BW:FcγRI-ζ cells. (<b>C</b>) MRC-5 cells were infected with HCMV HB5ΔIRLΔgp68/Δgp34 (2 PFU/cell) for 72 h before soluble proteins were added in graded concentrations concomitantly with BW:FcγRIIIA-ζ responder cells. MRC-5 fibroblasts were opsonized with 1∶50 diluted Cytotect for 30 min. After removing of unbound antibodies by washing, soluble proteins and BW:FcγR-ζ transfectants were added and co-cultivated overnight. (<b>D</b>) as in (C), but HCMV HB5ΔIRLΔgp68/Δgp34 infected target cells were opsonized with 1∶10 diluted Cytotect and BW:FcγRIIA-ζ cells were used as responders. n = 3 replicates, means with standard deviations (error bars) are shown for 2 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

Viral FcγRs bind Fcγ on the cell surface and opsonizing IgG dependent FcγR activation is restricted to the late phase of HCMV but not HSV replication.

<p>(<b>A</b>) MRC-5 cells were infected with 2 PFU/cell for 72 hpi with HCMV HB5 wildtype, HB5Δgp68, HB5ΔIRLΔgp34 or HB5ΔIRLΔgp68/Δgp34 (left) or 24 hpi with HSV-1 (right). Cells were resuspended with PBS/2% (vol/vol) FCS containing 2 mM EDTA, stained with hFcγ-FITC and measured in a FACS Canto II. Dead cells were excluded by PI or DAPI-staining. (<b>B</b>) MRC-5 cells were infected with 2–3 PFU/cell of HCMV HB5 wt or (<b>C</b>) HSV-1 strain F. After centrifugation enhancement of infection, cells were incubated 3 h at 37°C at 5% CO₂. After washing once, PAA (250 µg/ml) in D-MEM 10% (vol/vol) FCS was added. 48 hpi for HSV or 72 hpi for HCMV, cells were opsonized with grading dilutions of Cytotect for 30 minutes, washed twice with D-MEM 10% (vol/vol) FCS and co-cultivated with 1×10⁵ BW:FcγR-ζ reporter cells per well. Measurement of mIL-2 in supernatants after 16 h of co-cultivation of reporter cells with targets was performed by ELISA. Values are presented in the graphic as OD 450 nm. 3 independent wells were measured, means are shown with standard deviations (error bars) for 2 independent experiments. Significance of results (Student's t-test) is presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001.</p

HSV-1 gE, HCMV gp68 and HCMV gp34 interfere with host FcγR activation upon opsonization of cells with polyclonal immune IgG.

<p>(<b>A</b>) The HSV vFcγR gE inhibits FcγRIIIA and FcγRIIA activation but fails to inhibit FcγRI. Human MRC-5 fibroblasts were infected with 2 PFU/cell of HSV-1 strain F wt and ΔgE for 24 h. Cells were opsonized with Cytotect at different concentrations for 30 min. After removing of unbound antibodies with D-MEM 10% (vol/vol) FCS, 1×10⁵ BW:FcγR-ζ transfectants per well were added and co-cultivated overnight. BW:FcγR activation was determined by measuring mIL-2 by ELISA. Three independent replicates were measured; means with standard deviations (error bars) are shown for 4 independent experiments. (<b>B</b>) HCMV vFcγR gp68 interferes with FcγRIIIA, FcγRIIA and FcγRI activation. MRC-5 cells were infected with HCMV HB5 wt virus or HB5Δgp68 (2 PFU/cell) for 72 h. Fibroblasts were opsonized with Cytotect at different concentrations for 30 min. After removing of unbound antibodies by washing, 1×10⁵ BW:FcγR-ζ transfectants were added per well. Measurement of mIL-2 in supernatants after 16 h of co-cultivation of reporter cells with targets was performed by ELISA. Values are presented in the graphic as OD 450 nm. Three independent wells were measured; means with standard deviations (error bars) are shown for 4 independent experiments. Significance of results (Student's t-test) are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004131#ppat.1004131.s006" target="_blank">Table S1</a> as *: p<0.05 **: p<0.01 ***: p<0.001. (<b>C</b>) HCMV vFcγR gp34 interferes with FcγRIIIA, FcγRIIA and FcγRI activation. As in (B) but MRC-5 cells were infected with HCMV HB5ΔIRL, HB5ΔIRLΔgp34 or HB5ΔIRLΔgp68/Δgp34 (2 PFU/cell) for 72 h. (<b>D</b>) gp34 and gp68 interfere with FcγR activation in AD169varL infected cells. As in (B) but MRC-5 cells were infected with AD169varL wt, AD169varLΔgp68, AD169varLΔgp34 or AD169varLΔgp68/Δgp34.</p

The HCMV vFcγRs gp68 and gp34 bind antigen-IgG complexes.

<p>(<b>A</b>) Schematic representation of the ‘antibody bipolar bridging’ model. (<b>B</b>) Lysates of SKOV-3 cells containing the heterocomplex of vFcγR-FLAG, antibody and antigen are immunoprecipitated using an anti-FLAG agarose. SKOV-3 cells expressing HER2 antigen on the surface were infected with rVACV expressing the vFcγRs before opsonized with the trastuzumab antibody (bipolar bridging-antibody) (T) or an isotype control IgG1 antibody, palivizumab (P). Lysates were prepared after incubation of infected cells with antibody. An anti-Flag agarose IP was performed and retrieved antigens were detected in western blot with anti-ErbB2-specific mAb recognizing human HER2, anti-human IgG, and anti-Flag (M2, Sigma-Aldrich) detecting the Flag-tagged vFcγRs. Equal expression of HER2 in cell lysates was verified by western blot analysis with an anti-ErbB2-specific rabbit mAb which detects human HER2 (bottom).</p