Human Cytomegalovirus Fcγ Binding Proteins gp34 and gp68 Antagonize Fcγ Receptors I, II and III

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.


Introduction
Human cytomegalovirus (HCMV) constitutes the prototypical human pathogenic b-herpesvirus found worldwide with high immunoglobulin G (IgG) seroprevalence rates of 50-98% [1]. Despite the expression of a very large antigenic proteome of approximately 750 translational products [2], HCMV avoids sterile immunity and invariably persists lifelong in the human host in a latent state with periodic phases of reactivation and virus shedding. While infection of immune competent individuals is usually subclinical, HCMV causes severe symptoms in immunocompromised individuals and congenitally infected newborns [1,3]. Cytomegalovirus immune control is organized in a hierarchical as well as redundant manner, with crucial roles for natural killer (NK) cells as well as T lymphocytes [4]. HCMV expresses a large set of immune evasion genes that impair recognition of infected cells by CD8+, CD4+ and NK effector cells and thus facilitate virus persistence, spread and superinfection [5][6][7] while cellular immune responses are nevertheless indispensable for CMV immune surveillance. Experimental and clinical evidence suggest that cytomegalovirus can persist for the lifetime by effectively defending itself from both cellular and humoral immunity. In the absence of either viral immune evasion genes or subsets of immune cells, the balance of pathogenesis versus clearance of the virus can be tilted. For example, B cell deficient mice exhibit a much higher susceptibility during recurrent mouse cytomegalovirus (MCMV) infection compared to control mice, reflected by 100-1,000-fold increased titers in the absence of CMV-specific IgG [8]. Moreover, adoptive transfer of memory B cells into naïve Rag 2/2 mice is sufficient for long term protection from lethal MCMV disease [9], and passive immunization with immune IgG reduces MCMV-induced pathology in newborn mice [10]. In clinical settings, HCMV-immune IgG preparations are used with varying degrees of success. Human intravenous hyperimmune immunoglobulin against HCMV (e.g. Cytotect) significantly lowers the risk of congenital CMV infection and disease at birth when given to primary HCMV-infected pregnant women [11]. Nevertheless, meta-analyses of clinical studies with solid organ transplant recipients as well as patients undergoing hematopoietic stem cell transplantation document little if any benefit of IgG prophylaxis against HCMV disease [12][13][14].
IgG antibodies have two functional domains: the fragment antigen binding (Fab) that contains the paratope recognizing the respective epitope of the antigen and the fragment crystallisable (Fc) which recruits IgG effector functions. Receptors for the Fc domain of IgG (FccRs) are expressed on immune cells to connect the humoral and cellular branches of immunity. Upon IgG binding and receptor activation, FccRs trigger a diversity of effector responses including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, endocytosis of immune complexes and cytokine production. Importantly, the set of human FccRs includes different activating members, i.e. FccRI (CD64), FccRIIA (CD32A), FccRIIC (CD32C) and FccRIIIA (CD16) which differ in immune cell distribution, affinity for distinct IgG subclasses [15] and effector functions elicited upon activation [16][17][18][19].
Fcc binding activity on the surface of HCMV-infected cells has long been reported [20], but the consequences of Fcc binding to immune responses are unknown [21]. The HCMV Fcc binding proteins gp34 and gp68 are type I transmembrane glycoproteins encoded by independent genes, RL11 and UL119-UL118, respectively, which are fully dispensable for HCMV replication in vitro [22,23]. Both HCMV proteins show cell surface disposition and exquisite ligand specificity for human IgG but no other Ig classes (e.g. IgA or IgM) [22]. Minimal sequence relatedness in their extracellular domains with particular immunoglobulin supergene family domains present in FccRI and FccRII/III suggests differing binding characteristics from those of host FccRs [22]. In contrast to host FccRs, both gp34 and gp68 recognize Fcc in a manner independent of N-linked glycosylation, further corroborating a binding mode to Fcc which is distinguishable from host FccRs [24].
Cytomegaloviruses frequently reactivate and can super-infect despite the presence of relatively high levels of HCMV-specific IgG [25][26][27] which raises the apparent question by which mechanism HCMV avoids antibody-mediated immune control. One conceivable possibility is that HCMV-encoded FccRs gp34 and gp68 compete with cellular FccRs. To test this hypothesis, we had to established a suitable FccR activation assay that allows a comprehensive analysis of the activation status of individual FccRs [28]. We compared cells infected with viruses lacking gp34 and/or gp68 upon opsonization with graded doses of polyclonal HCMVimmune IgG. As a control, we included the human a-herpesvirus herpes simplex virus 1 (HSV-1), which expresses a viral FccR composed of the glycoproteins gE and gI that is known to protect infected cells from ADCC elicited by HSV-immune IgG [29,30]. Our approach identifies FccRI, FccRIIA and FccRIII as principal targets of both HCMV gp34 and gp68, while the prototypic HSV-1 FccR gE was found to inhibit only FccRIIA and FccRIII. This is the first experimental proof of CMV-encoded glycoproteins interfering with IgG-mediated immunity.

HCMV and HSV-1 encoded viral FccRs (vFccRs) bind Fcc on the surface of infected cells
To assess the relative surface density of viral Fcc receptors on the plasma membrane of HSV and HCMV-infected cells, Fcc binding was evaluated by flow cytometry using FITC-labeled Fcc fragment. As expected, Fcc-FITC surface binding was observed for HSV wt virus-and the gE revertant virus-infected cells, but not for cells infected with DgE HSV ( Figure 1A). MRC-5 cells infected with either of two HCMV HB5 single vFccR deletion mutants, HB5Dgp68 or HB5DIRLDgp34 [22], were decorated at the cell surface with diminished levels of Fcc-FITC compared to wt-infected control cells ( Figure 1A). Cells infected with a HCMV mutant lacking both gp68 and gp34 [31] showed only very low Fc-binding when compared to mock-infected fibroblasts ( Figure 1A). Together with our previous experiments documenting Fcc binding upon ectopic expression of gp34 and gp68 using recombinant vaccinia viruses (rVACV) [22] these data define gp34 and gp68 to be sufficient and essential for Fc binding by HCMVinfected cells.
FccR activating IgG almost exclusively recognizes antigens expressed during the late phase of HCMV replication In productively infected cells, herpesvirus gene expression is regulated in a cascade fashion. Viral proteins encoded by genes of the early phase of infection are required for viral DNA replication, which is a prerequisite for the subsequent expression of structural virion proteins during the late phase of gene expression. To assign the immune-dominant HCMV and HSV surface antigens recognized by opsonizing IgG to the temporal class of genes, we applied the novel reporter cell system allowing quantification of host FccR activation [28]. This assay is based on co-cultivation of antigen-bearing cells with reporter cells stably expressing FccR-f chain chimeric receptors which produce mouse IL-2 upon recognition of immune IgG, provided that the opsonizing antibody is able to activate the particular FccR [28]. Importantly, BW5147:FccR-f reporter cells are neither activated by cells lacking the appropriate antigen (e.g., non-infected cells) nor by antigen-expressing cells which had been cultivated in absence of IgG or in presence of non-immune IgG, proving strict antigen-

Author Summary
Herpes viruses persist lifelong continuously alternating between latency and virus production and transmission. The latter events occur despite the presence of immune IgG antibodies. IgG acts by neutralization of virions and activation of immune cells bearing one or more surface receptors, called FccRs, recognizing the constant Fc domain of IgG. Activating FccRs induce a wide range of immune responses, including antibody dependent cellular cytotoxicity (ADCC) of virus-infected cells by natural killer (NK) cells, cytokine secretion and the uptake of immune complexes to enhance antigen presentation to T cells. We demonstrate that the HCMV glycoproteins RL11/gp34 and UL119-118/gp68 block IgG-mediated activation of FccRs. A novel reporter cell-based assay was used to test FccRs individually and assess their relative susceptibility to each antagonist. This approach revealed that gp34 and gp68 block triggering of activating FccRs, i.e. FccRI (CD64), FccRII (CD32A) and FccRIII (CD16). Co-immunoprecipitation showed the formation of ternary complexes containing IgG, IgG-bound antigen and the viral antagonists on the cell surface. Assigning the redundant abilities of HCMV to hinder IgG effector responses to the viral Fc binding proteins, we discuss gp34 and gp68 as potential culprits which might contribute to the limited efficacy of therapeutic IgG against HCMV.
and immune IgG specificity of the assay ( Figure S1 and Reference [28]). Late phase gene expression was blocked using 250 mg/ml phosphonoacetic acid (PAA) which blocks the viral DNA polymerase and is the active component of the clinically approved anti-HCMV drug Foscarnet. At 72 and 48 hpi, resp., infected cells were opsonized with graded concentrations of intravenous immunoglobulin (IVIG) Cytotect as a source of human HCMV-and HSV-immune IgG. As expected, immune IgG did not induce receptor activation (IL-2 response) in the presence of mock-infected cells ( Figure 1B). While late antigens of HCMV efficiently triggered FccRIII reporter cells, infected cells arrested in the early phase of replication elicited very poor if any responses ( Figure 1B). On the contrary, early antigens of HSV-1 were sufficient to efficiently trigger FccRIII and HSV late antigens  only slightly increased FccR-f activation ( Figure 1C). Despite the fact that Cytotect is prepared from donors selected for particularly high HCMV IgG titers [11,32] we found -in agreement with earlier studies [28] -that Cytotect contains higher titers of HSVimmune IgG activating FccRIII compared to FccRIII-reactive HCMV-immune IgG. Due to this fact, IVIG dilutions used for HSV-1 and HCMV experiments had to be chosen differently. The relatively poor responses triggered by IgG opsonized HCMVinfected cells was compatible with our hypothesis that HCMV could be able to reduce the activation of FccRIII by immune IgG, and that vFccR gp68 and gp34 might represent candidates for such inhibition.
Interference with host FccR activation by HSV-1 gE, HCMV gp68 and HCMV gp34 To test the conjecture that vFccR gp68 and gp34 could prevent host FccR activation by HCMV IgG, we pursued the BW5147:FccR-f reporter cell approach. To assess if and to what degree viral FccRs can interfere with host FccRs activation, we first compared responses of FccRIII reporter cells co-cultured with IgG-opsonized HSV-1 wt infected vs. HSV-1 DgE-infected MRC-5 cells (Figure 2A). Inhibition of ADCC by PBMCs was reported to be a function of the prototypic HSV-1 FccR gE [30], albeit the specific host FccRs which are blocked by gE have not been elucidated yet. As before ( Figure 1C), we observed a dosedependent activation of the reporter cells upon co-cultivation with HSV-1-infected cells but not with mock infected cells. The DgE HSV-1 mutant led to an increased activation of FccRIIIA upon opsonization of target cells with Cytotect, in accordance with published data [29,30]. The same type of result was obtained when using BW5147:FccRIIA-f reporter cells, indicating that gE also antagonizes activation of this host Fcc receptor. Surprisingly, gE enhanced, rather than inhibited the IgG-dependent activation as deduced from the overall superior activation of the chimeric FccRI-f by wt HSV-1 when compared to HSV-1 DgE (Figure 2A).
The results of HSV-1 gE encouraged us to subsequently test HCMV HB5Dgp68 and HB5DIRLDgp34 mutants using the same experimental strategy. MRC-5 fibroblasts were left uninfected or infected with wt-HCMV strain HB5 versus HB5Dgp68 ( Figure 2B), or with HB5DIRL (the parental virus of the following mutants) versus HB5DIRLDgp34 and HB5DIRLDgp68/ Dgp34, resp., a double mutant lacking both gp68 and gp34 ( Figure 2C). 72 h post HCMV infection, target cells were incubated with graded dilutions of Cytotect before BW:FccRIIIAf responder cells were added. HB5Dgp68 induced a clearly higher response over an extended range of IgG dilutions compared to wt-HCMV HB5 infected cells. Likewise, HB5DIRLDgp34-opsonized cells induced clearly higher reporter cell responses compared with HB5DIRL opsonized targets, while HB5DIRLDgp68/Dgp34 exhibited only marginal further increase of the response ( Figure 2C). These results suggested that cells infected with virus mutants lacking viral Fc-binding proteins elicit exaggerated activation of FccRIIIA, provided that the amount of opsonizable HCMV antigens is indeed comparable between the viruses analyzed. To verify this supposition, cells were labelled with F(ab) 2 antibody fragments prepared from Cytotect and analysed by FACS. As shown in Figure S2, cells infected with HCMV mutants lacking gp34 and/or gp68 did not show higher levels of opsonizing antigens on the plasma membrane. To compare the relative impact of gp68 versus gp34 on FccRIII activation with a higher degree of accuracy, i.e. in the context of an identical HCMV genome possessing a preserved UL/b9 gene region, another set of targeted vFccR gene deletions was constructed based on the AD169varL derived BACmid pAD169 which carries unlike pHB5 only a single copy of TRL genes including TRL11 [33]. As demonstrated in Figure 2D, targeted deletion of UL119-118/gp68 and TRL11/gp34 reproduced the increased activation of BW:FccRIIIA-f responder cells, while combined deletion of both vFccRs only marginally enhanced the response further. Next we determined if gp34 and gp68 could affect further activating host FccRs and performed co-cultivation assays with MCR-5 cells infected with the same panels of HCMV mutants after opsonization with Cytotect and incubated with BW:FccRIIA-f and BW:FccRI-f reporter cells ( Figure 2C-D). While deletion of both HCMV Fcc-binding proteins resulted in significantly enhanced responses by both FccRIIA and FccRI, the isolated removal of gp68 resulted in a slightly more drastic phenotype with regard to BW:FccRIIA-f activation ( Figure 2D). Notably, combined removal of gp34 and gp68 led to a Dgp34-like phenotype, contrasting to the additive effect seen with FccRIII at low IgG concentrations. In conclusion, the data suggested that both of the HCMV-encoded FccRs might have developed the ability to interfere with the activation of FccRIII, FccRIIA and FccRI, while HSV gE blocks FccRIII and FccRIIA but fails to inhibit FccRI activation.
UL118-119 and RL11 gene reversion restore resistance to FccR activation by immune IgG To exclude the possibility that second site mutations which occurred during the BACmid mutagenesis procedure are responsible for the observed loss of HCMV-mediated inhibition of host FccR activation by immune IgG, an entirely independent panel of virus deletion mutants and the appropriate rescued versions were generated. The mutants were constructed using the HCMV TB40/E-derived BACmid [34] taking advantage of i) a single gene copy of RL11 coding for gp34, ii) a complete HCMV ULb9 gene region lacking in HCMV HB5 but present in HCMV clinical isolates and iii) a technically more feasible re-insertion strategy of the vFccR coding genes. MRC-5 fibroblasts were left uninfected or infected with the HCMV TB40/E wt expressing gp68 and gp34, or with gp68 and gp34 single gene deletion mutants, resp., or independent single gene revertant mutants expressing gp68 or gp34. Using BW:FccRIIIA-f responder cells and graded concentrations of HCMV immune IVIG, the gp34 and gp68 TB40/E deficient mutants elicited a stronger FccR-f activation response than the TB40/E wt ( Figure S3A), while the density of opsonizing cell surface antigens was not altered ( Figure S3B). The finding that three independent virus mutants lacking Fc binding proteins show congruent phenotypes makes unintended second site mutations as cause for the effect highly unlikely. Nevertheless, revertant viruses were assessed. As expected, both of the revertant viruses exhibited a wt-like phenotype ( Figure S3A). In comparison to HCMV HB5, HCMV TB40/E shows a more protracted replication kinetic. Consistently, we observed more efficient IgG-dependent activation of FccRIIIA-f at 96 hpi compared with 72 hpi. Therefore, HCMV TB40/E-based assays were performed 96 h post infection. The HCMV TB40/E results confirmed that both HCMV-encoded FccRs inhibit the activation of FccRIIIA and that their reinsertion into the virus genome reestablishes the vFccR inhibition phenotype.

Inhibition of IgG1 (trastuzumab) mediated activation of FccRs
To test if gp34 and gp68 suffice to impair IgG-dependent activation of FccRs, two factors of our experimental approach were modified: (i) gp34 and gp68 were expressed outside the context of  Figure 3A). Opsonized VACV-infected cells exhibited a reduced capacity to trigger FccRIIIA in comparison to mock cells, most likely due to the protein host shut-off function of VACV. Importantly, trastuzumabmediated FccRIIIA triggering was further impaired by rVACV gE, providing proof of principle that ectopically expressed gE suffices to interfere with IgG1-dependent FccRIII activation. In contrast to FccRIII, trastuzumab reproducibly failed to induce FccRII responses ( Figure 3A). When trastuzumab-opsonized cells were probed with FccRI transfectants, the presence of gE did not attenuate but rather enhanced the response ( Figure 3A), confirming the unexpected phenotype in the HSV-infected cell setting observed before ( Figure 2A). Next, rVACVs were used to express gp34 and gp68 ectopically in HER2 positive SKOV-3 targets which were opsonized with different concentrations of trastuzumab before co-culture with the same panel of responder cells as already described ( Figure 3B). Both gp34 as well as gp68 significantly reduced activation of FccRIII and FccRI, albeit in this setting gp34 seemed slightly more potent than gp68. In summary, deploying a gain-of-function approach and using a monoclonal human IgG1, the results verified that both HCMV FccRs are sufficient to prevent the activation of FccRI and FccRIII.

Interference with host FccRIIA activation by ectopic expression of herpesviral FccRs
Trastuzumab is not capable to activate FccRIIA (see above, Figure 3A-B). Nevertheless, we wished to assess the effect of ectopically expressed vFccRs on FccRIIA activation. Therefore, in a further approach CD20 transfected 293T cells [35] were infected with rVACV expressing gE, gp68 or gp34 before opsonized with rituximab another well-defined humanized therapeutic monoclonal IgG1 antibody ( Figure S4A and S4B). All vFccRs inhibited FccRIIA activation verifying that ectopic expression of the viral Fcc binding proteins gE, gp34 and gp68 hinder the activation of the host FccRIIA in a gain-of-function approach.

FccRIII inhibition by gp34 and gp68 across IgG subclasses
Humans respond to HCMV infection with the production of IgG1 which is the immunodominant subclass, followed by IgG3, 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 Table  S1 as *: p,0.05 **: p,0.01 ***: p,0.001. (C) HCMV vFccR gp34 interferes with FccRIIIA, FccRIIA and FccRI activation. As in (B) but MRC-5 cells were infected with HCMV HB5DIRL, HB5DIRLDgp34 or HB5DIRLDgp68/Dgp34 (2 PFU/cell) for 72 h. (D) gp34 and gp68 interfere with FccR activation in AD169varL infected cells. As in (B) but MRC-5 cells were infected with AD169varL wt, AD169varLDgp68, AD169varLDgp34 or AD169varLDgp68/ Dgp34. doi:10.1371/journal.ppat.1004131.g002  Table S1 as *: p,0.05 **: p,0.01 ***: p,0.001. doi:10.1371/journal.ppat.1004131.g003 while HCMV-immune IgG2 and IgG4 is detected only at very low levels if produced at all [36,37]. In contrast to HSV-1 gE, HCMV gp68 and gp34 bind monomeric IgG of all human subclasses, i.e. IgG1, IgG2, IgG3, and IgG4 [22], whereas gE does not bind IgG3 [38,39]. To assess whether HCMV gp68 and gp34 can inhibit FccRIIIA/CD16 activation through immune complexes formed by different IgG isotypes, we took advantage of a panel of rituximab-derived isotypic IgG antibodies. CD20 transfected 293T target cells [35] were infected with VACV wt or rVACV expressing gp68, gp34 or MULT-1 as a negative control and opsonized with anti-hCD20 IgG isotypes including an IgA constant region fused to the variable region of rituximab as an antibody control. CD20 expression revealed very similar levels of antigen expression on the cell surface of VACV target cells (data not shown). While opsonized IgG1 and IgG3 isotypes efficiently activated FccRIIIA, very little to no activation was observed with IgG2, IgG4 and IgA, confirming previous data [28]. Both gp34 as well as gp68 strongly reduced activation of FccRIIIA by IgG1 and IgG3 ( Figure 4). The data documented the inhibitory potency of both HCMV FccRs against IgG1 and IgG3-formed immune complexes and confirmed the functional distinction of gp34 and gp68 against HSV gE.

The herpesviral FccRs inhibit antibody dependent NK cell degranulation
CD16/FccRIII is an essential IgG receptor for activation of NK cells mediating ADCC responses [40,41] but also found on human cd T cells induced by HCMV infection [42]. The data obtained with the FccRIII-f reporter cells strongly suggested that gp34, gp68 as well as HSV-1 gE operate as inhibitors of FccRIII/CD16+ NK cells since BW:FccRIII-f responses showed an excellent match with CD107a mobilization of primary human NK cells upon CD16/ FccRIII cross-linking [28]. Therefore, we tested the activation of primary human NK cells by fibroblasts infected with HSV-1, HCMV and mutants devoid of viral Fcc binding proteins, respectively, in the presence of virus-immune opsonizing IgG in a CD107a degranulation assay [43]. The sources of the opsonizing IgG were sera donated by HSV/HCMV-seropositive donors ( Figure 5A and Figure 5B, resp.) or Cytotect ( Figure 5C). rhIL-2 overnight preactivated NK cells from HSV/HCMV sero-negative donors were enriched by negative selection and analyzed after 4 hours of co-incubation with infected cells opsonized with graded concentrations of immune IgG. HCMV encodes numerous inhibitors of NK cell activation [44,45]. To focus on IgG-dependent NK cell activation, NK activation was calculated and depicted as percentage of IgG-specific CD107a mobilization (i.e. percentage of CD107a-positive cells obtained with the immune antibody opsonizing target cells minus the percentage of CD107a-positive cells obtained with non-immune antibody treated target cells). A higher ratio of IgG-dependent CD107a positive cells in the case of HSV-1 DgE-infected cells compared with wt HSV-1 infected cells was observed ( Figure 5A). Likewise, the HB5Dgp68, HB5DIRLDgp34 and HB5DIRLDgp68/Dgp34 HCMV mutants yielded clearly increased IgG-dependent CD107a mobilization as HCMV HB5 ( Figure 5B). As observed with BW:FccR-f responder cells, gp34 and gp68 inhibited FccRIIIA NK activation independently, but no additive effects were noted upon deletion of both vFccRs. To exclude donor-specific effects, NK cells from six different donors were analyzed in degranulation assays comparing HCMV HB5 wt with HB5DIRLDgp68/Dgp34 -infected targets opsonized with Cytotect as a source of immune IgG and non-immune sera as a negative control. All donors showed a higher percentage of IgGdependent CD107a positive cells in the case of HB5DIRLDgp68/ Dgp34 -infected cells ( Figure 5C). Taken together, these data demonstrated that vFccR gE, gp34 and gp68 on the surface of infected cells mediate inhibition of IgG-dependent NK cell degranulation.
HCMV FccRs form ternary complexes with antigen and IgG on the cell surface compatible with antibody bipolar bridging HCMV gp68 was found to bind the Fc C H 2-C H 3 interface of monomeric IgG at nanomolar affinity [24]. To get insight into the intermolecular interactions underlying the inhibitory function of gp34 and gp68 when blocking antigen-antibody complexes, we tested the occurrence of a physical complex on the surface of cells consisting of the target antigen, bound IgG and each of the HCMV FccRs. We took advantage of immune complexes (composed of trastuzumab and its antigen HER2) which were shown to be sensitive to the blockade through gp34 and gp68 when activating FccRIII and FccRI ( Figure 3B). HER2expressing SKOV-3 cells were infected with rVACV expressing Flag-tagged gp34, gp68 or a control protein, DIg1-m138, a nonfunctional MCMV m138/fcr-1 truncation mutant [46] and opsonized with trastuzumab ('T') or with an IgG1 isotype control antibody, palivizumab ('P') (see sketch in Figure 6A). VACVinfected cells were thoroughly washed to remove unbound antibodies and subsequently lysed. To exclude Fcc-mediated binding of vFccRs through anti-Flag antibodies, vFccR proteins were immunoprecipitated using a-Flag F(ab) 2 -coupled agarose beads. The precipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting using an HER2 specific antibody ( Figure 6B). An anti-human IgG-specific antibody was used to detect the co-precipitated antibody. An immuno blot confirmed expression and immunoprecipitation of the vFccRs. Retrieval of palivizumab by gp34 and gp68 was weaker than retrieval of trastuzumab. This difference could be explained by the fact that trastuzumab could be retained by the cells via vFccRs and via HER2, while palivizumab could only be retained by vFccRs. Subsequently, gp34 and gp68 retrieved trastuzumab antibodies bound to HER2 during lysis and precipitation. Nevertheless, coprecipitation of human HER2 molecules occurred only in the presence of specific antibody trastuzumab and the HCMV FccRs but not in the negative control DIg1-m138 ( Figure 6B, lanes 5, Figure 4. HCMV gp68 and gp34 inhibit FccRIII activation by rituximab antibody isotypes. CD20 transfected 293T cells were infected for 16 h with 2 PFU/cell of VACV wt or rVACV expressing gp68, gp34 or MULT-1 as a control. After opsonization with 1 mg/ml of each antibody isotype for 30 min. and removing of unbound antibody by washing, cells were co-cultivated with 1610 5 BW:FccRIIIA-f 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 Table S1 7 and 3, respectively). Binding of human IgG antibodies, trastuzumab and the isotype control antibody palivizumab, was observed to both vFccRs ( Figure 6B, lanes 5, 6, 7 and 8). No binding of trastuzumab or palivizumab was detectable to the DIg1-m138-Flag protein ( Figure 6B, lanes 3 and 4). Taken together, the ability of cell surface resident vFccRs gp34 and gp68 to bind to IgG immune complexes was demonstrated. This finding is compatible with the model of ''antibody bipolar bridging'' described for the HSV-1 FccR gE [47][48][49]. According to this concept, epitope-bound IgG on the surface of a virus-infected cell is simultaneously sequestered by the gE:gI complex via its Fc domain, thus preventing the activation of immune effector molecules via host FccRs.

Soluble ectodomains of HCMV vFccRs inhibit IgGdependent host FccRs activation
Soluble truncation versions of HSV-1 gE and HCMV gp68 were instrumental to unravel structural requirements and stoichiometry of herpesviral FccRs forming complexes with Fcc [24,49,50]. To test whether membrane insertion of gp34 and  gp68 is required to interfere with the activation of host FccRs, recombinant C-terminally truncated ectodomains of HCMV FccRs were generated and purified from supernatants of transfected human 293 cells. To evaluate if soluble gp34 (sgp34) and soluble gp68 (sgp68) are sufficient to block triggering of host Fcc receptors, HER2-positive cells were opsonized with trastuzumab and different amounts of recombinant soluble vFccRs were concomitantly added to BW:FccRIIIA-f cells ( Figure 7A) or BW:FccRI-f cells ( Figure 7B). Soluble ICOS ligand (sICOSL) served as a negative control protein. Both sgp34 and sgp68 were able to inhibit activation of the reporter cells expressing FccRIIIA, although clear differences in concentration dependency between soluble vFccRs were observed. In contrast to full-length HCMV FccRs, sgp34 was more potent against FccRIIIA compared to sgp68, since trace amounts of sgp34 hardly detectable in western blot ( Figure S5) were sufficient for significant inhibition. In the case of FccRI, sgp68 was not significantly reducing its activation by trastuzumab, suggesting the specific requirement of the gp68 transmembrane domain for effective inhibition of FccRI/CD64.
To extend the data to HCMV infection and to test BW:FccRIIA-f cells, a polyclonal antibody preparation (IVIG, Cytotect) was used to opsonize MRC-5 fibroblasts infected with the HCMV HB5DIRLDgp68/Dgp34 mutant lacking both gp68 and gp34. Using BW:FccRIIIA-f reporter cells, both of the soluble HCMV vFccRs prevented activation when compared with treatment of cells with the sICOSL control ( Figure 7C). Moreover, this approach allowed to test activation of BW:FccRIIA-f cells which did not respond to trastuzumab (see Figure 3). As depicted in Figure 7D, FccRII responses were also sensitive to sgp34 and, to a lesser extent, sgp68. These results provide proof of principle that soluble HCMV FccRs retain FccR blocking abilities, and that sgp34 is particularly efficient.

Soluble ectodomains of HCMV vFccRs inhibit IgGdependent NK cell degranulation
In an attempt to extend the previously made observation of sgp34 and sgp68-mediated inhibition of IgG-triggered FccRIII/ CD16+ BW5147 responder cells to primary human NK cells, purified IVIG Cytotect was coated directly to a plate serving as a source of 'immune-complexed' IgG. After blocking with D-MEM 10% FCS (vol/vol), soluble proteins, IL-2 pre-activated primary NK cells and a-CD107a-PECy5 antibody were added. Soluble ICOS ligand (sICOSL) served as a negative control protein. In the absence of coated IgG, only 10% of NK cells responded with CD107a mobilization, while in the presence of coated IgG more than 70% of NK cells translocated CD107a to the cell surface ( Figure 8A), confirming the IgG-dependency of the elicited NK cell response. Importantly, both sgp34 and sgp68 were able to  Table S1 as *: p,0.05 **: p,0.01 ***: p,0.001. doi:10.1371/journal.ppat.1004131.g007 inhibit IgG-dependent NK degranulation, although a clear difference in the concentration dependency between soluble vFccRs was observed. Consistent with the results received with BW:FccRIIIA-f reporter cells ( Figure 7C), sgp34 was more potent against IgG-dependent NK activation compared to sgp68, since trace amounts of sgp34 ( Figure 8B) were sufficient to significantly interfere with degranulation of NK cells. Using a gain of function approach the data confirmed the inhibitory capacity of gp34 and gp68 to attenuate IgG-mediated NK cell activation.

Discussion
Here we identified various members of the human FccR family, i.e. FccRI/CD64, FccRII/CD32A and FccRIII/CD16A, to be targeted by the HCMV FccRs gp34 and gp68 which act as antagonists of ligand induced FccR responses. This ability enables HCMV to evade from IgG effector responses and should have direct proviral effects in scenarios of post-acute and recurrent infection when glycoprotein-specific IgG antibodies are synthesized [51]. Several independent experimental approaches support this conclusion: i) HCMV HB5-derived mutants with deletions of the gp34 and gp68 coding genes, TRL11/IRL11 and UL118-119, respectively, showed significantly increased activation of host FccRs upon opsonization of infected cells with polyclonal HCMV immune IgG using different types of responder cells (i.e. BW5147 transfectants expressing FccR-f chain chimeras and primary human NK cells); ii) this HCMV phenotype was reproduced with targeted RL11 and UL118-119 mutants of the AD169varL and TB40/E strain and iii) fully reversed by retransfer of the responsible genes into the RL11and UL118-119-deficient TB40/E genomes; iv) a gain-of-function approach based on ectopic VACV-based expression of gp34 and gp68 which allowed functional analysis of well characterized therapeutic human monoclonal antibodies and different IgG isotypes thereof and v) functional testing of recombinant soluble ectodomains of both HCMV inhibitors using BW5147:FccR reporter cells as well as primary human NK cells. Importantly, the inhibitory effect of gp34 and gp68 was demonstrated at physiological concentrations of polyclonal HCMV immune IgG, i.e. within an extended concentration range of human serum and ten to fifty fold lower.
The quite complex and overlapping expression patterns of host Fc-IgG receptors on a multitude of diverse human immune cell (sub-)populations [17] have obstructed a systematic functional analysis of individual host FccRs which differ with respect to molecular and functional features including the composition of their ectodomain, intracellular signaling and IgG subclass preferences. Only a recently developed methodologically broadly tested and proven reporter cell assay [28] enabled a comprehensive and quantitative functional assessment of potential viral antagonists and their relative effectiveness against distinct host FccRs. The new methodology was complemented and validated by immunological as well as biochemical assays, i.e. the use of primary human NK cells as natural responder cells and immunoprecipitation studies, the results of which accord very well with the findings made with the BW5147:FccR-f test system. Last but not least, the well-known viral FccR inhibitor, HSV gE, was included as an internal control. Previous publications reported inhibition of ADCC, virion neutralization and complement mediated virolysis by HSV gE [47,52,53]. Since the blockade of ADCC by gE was not yet attributed to a specific host FccR [30,47,54], analysis of the relative impact of HSV gE on distinct host FccRs represents a novel aspect of our study. On the basis of the test performance of HSV gE, both gp34 and gp68 demonstrated an at least equivalent if not superior efficacy to block FccRIII and FccRIIA mediated responses. Surprisingly and contrasting with both HCMV vFccRs, gE enhanced rather than attenuated FccRI activation. This observation warrants further studies how HSV-infected cells affect FccRI bearing immune cells like monocytes, macrophages, DCs and neutrophils in the presence of HSV-immune IgG.

Structural requirements for FccR inhibition
The inhibition mechanism of IgG-mediated effector functions by gE has been suggested to involve 'antibody bipolar bridging' [47]. Pioneering studies of the Bjorkman laboratory demonstrated that the architecture of the gE/gI-IgG complex allows antibody bipolar bridging [49], whereby the gE binding site for Fcc does not directly overlap with the binding sites to the host FccRs or the C1q component of complement, which both bind to the upper hinge region of IgG or near the C H 2 domain [55,56]. Therefore, the structure of the gE/gI-Fc complex does not directly explain how gE binding to the Fc region of IgG leads to evasion from FccRand complement-mediated immune responses. Our biochemical data reveal formation of ternary heterocomplexes composed of antigen, IgG and gp34/gp68, i.e. a molecular configuration compatible with the minimal requirements of the concept of 'bipolar bridging' [47,49]. The observation that soluble gp34 and gp68 remain potent inhibitors of FccR activation demonstrates that the functional inactivation of the host FccR on the responder cell does not require fixation of the opsonized IgG to the plasma membrane as insinuated by the classical concept of bipolar bridging. Although there is no crystal structure available for any HCMV vFccR, detailed biochemical evidence was generated of how HCMV FccRs recognize Fcc, particularly for gp68. The gp68 Fcc binding site was mapped to the C H 2-C H 3 interface region of Fcc [24] which is remote from the FccRII/III contact site, that involves the hinge between the Fcc and Fab domains including the upper portion of the C H 2 domain [55,57]. Specifically, gp68 binding to Fcc is affected by mutations at the C H 2-C H 3 domain interface of IgG, mapping its binding site to determinants situated nearby but not identical with those that are recognized by gE [24]. We observed robust functional differences between HSV-1 gE and HCMV gp68 in their manipulation of FccRI, further substantiating the mechanistic differences between these viral inhibitors regarding their interaction mode with Fcc. While the binding of IgG by gE must induce conformational changes of the antibody that result in an enhancement of FccRI activation, gp68 binding to IgG induces the opposite effect. Since both gE and gp68 had concordant inhibitory effects on FccRIII, our data further imply that FccRI and FccRIII must bind IgG in a differential fashion. Importantly, the length and flexibility of the hinge region varies considerably among the IgG subclasses, and IgG3 differs from the other subclasses by its unique extended hinge region which is approx. four times as long as the IgG1 hinge, leading to the most hinge-mediated flexibility among human IgG subclasses [58]. Notably, we demonstrate efficient FccRIII blockade by IgG3-shaped immune complexes through gp68 and gp34 (Figure 4) which is not possible by HSV gE [36,37]. Thus, by analogy with HSV gE, HCMV gp34 and gp68 represent promising and unique tools to further probe into the diverse structural requirements of FccRI/II/III activation by immune complexes constituted by all IgG subclasses.

Functional redundancies of gp34 and gp68?
The presence of independent but redundant immunoevasins jointly targeting one particular immune control mechanism is a typical feature of cytomegaloviruses highlighting the antiviral power of the targeted immune component [6,[59][60][61][62]. At first glance, the HCMV FccR antagonists gp34 and gp68 exhibit a surprisingly similar effect on the whole range of activating host FccRs, despite their simultaneous synthesis during the early and late phase of HCMV replication [22]. As a consequence, removal of both inhibitors from the surface of HCMV infected cells did not reveal additive or even synergistic effects compatible with the notion that the two factors do not act in an obvious cooperative manner. This finding cannot be attributed to differences in the density of plasma membrane resident HCMV antigens between the HCMV gene deletion mutants compared in our study (see Figure S2 and S3). However, both of the antagonists could themselves represent antigens that are recognized by the F(ab) part of immune IgG, which could either directly activate host FccRs or block Fcc-mediated bridging of opsonizing IgG (as a counter defense of humoral immunity against vFccRs) and thus indirectly enhance host FccR triggering. Moreover, both of the HCMV FccRs may fulfill further proviral but Fcc-independent functions which exert separate pressures to adapt. This is exemplified by the MCMV m138/fcr-1 molecule which down-regulates the NKG2D ligands MULT-1, H60, RAE-e [46,63] as well as the B7-1 molecule CD80 [64] beyond its Fcc binding activity. In addition, besides gp34 and gp68 additional HCMV FccRs become expressed on infected cells (Mercé-Maldonado and Hengel, in preparation), one of which is encoded by RL13 [65]. Thus gp34 and gp68 may be part of a much more complex network of coexpressed HCMV FccRs jointly combating their host opponents, and removal of one player could confound their interplay and nested hierarchies. Next, drastic quantitative and qualitative differences in the potency of gp34 vs. gp68 became apparent when soluble molecules were compared. Thus it is tempting to speculate that shedding of vFccRs may be part of the molecular blueprint of particular vFccRs.

Which HCMV antigens elicit ADCC responses?
While there is extensive knowledge on antigens and processed epitopes which rule anti-HCMV T cell responses [66], viral antigens that are targets of ADCC dependent cellular immunity remain poorly defined. Our finding that late but not early antigens dominate the FccRIII/CD16 activating IgG response ( Figure 1B) appears a particular characteristic of HCMV when compared with HSV and could point to structural glycoproteins known to become exposed on the cell surface as the HCMV replication cycle progresses, e.g. gB [67], gH [68] and UL128 [69]. Guided by human antibodies with defined specificity, our BW5147-based FccR-f assay system could be instrumental to identify the relevant HCMV antigens and epitopes. In many tissues and organ compartments, including blood, HCMV is spreading intracellularly (e.g. via infected endothelial cells and leukocytes) rather than as free virions [70]. Therefore, ADCC-inducing IgG is plausible to represent a primary effective component of humoral immunity, which becomes only secondary attenuated by gp34 and gp68. Both immunoevasins could thus contribute to the relatively poor therapeutic efficacy of HCMV-immune IgG observed in a variety of clinical settings [12][13][14]. Thus a better knowledge of the optimal HCMV IgG epitopes on the one hand, and an understanding of the action of viral FccR antagonists on the other hand, could provide us with a basis for the targeted induction or even rational synthetic design of IgG molecules that allow an improved immunotherapy of HCMV diseases.
Infection of cells with HCMV and HSV was enhanced by centrifugation at 800 g for 30 min. If not stated otherwise, the cells were infected with 2-3 PFU/cell.

Human immunoglobulin preparations, human serum pools and humanized antibodies
A clinically used IVIG preparation [11] Cytotect [32,77] (batch no. A158024 and B797053, Biotest Pharma GmbH, Germany) containing ELISA reactive IgG specific for HCMV and HSV was used. For the FACS analysis of HCMV and HSV surface antigens, F(ab) 2 fragments were generated from Cytotect with the Pierce F(ab) 2 Micro Preparation Kit (Thermo Fisher Scientific Inc., Rockland, IL, USA) according to the manufacturer's instructions and controlled by Western Blot (data not shown). For the experiments with HCMV and HSV, a pool of two ELISA seronegative donors were used as a negative control. Trastuzumab was purchased from Genentech, Inc., USA and palivizumab from MedImmune, USA. The humanized anti-CD20 IgG1, IgG2, IgG3, IgG4 isotypes and IgA were purchased from InvivoGen, Toulouse, France. For the CD107a NK degranulation assay, an HCMV-and HSV-seropositive donor and a negative serum donor as sources of immune and non-immune IgG, respectively, were used. For proofing that the assay was antibody-antigen specific ( Figure S1), a pool of 6 HCMV-and HSV-seropositive donors and a pool of 2 negative serum donors as sources of immune and nonimmune IgG, respectively, were used. For serum preparation, blood was drawn from healthy volunteers after written informed consent.

Ethics statement
The experiments were approved by the Ethics Committee of the University Hospital Düsseldorf (no. 3410) in accordance with the Declaration of Helsinki. For serum and NK cells preparation, blood was drawn from healthy volunteers after written informed consent.

IgG dependent activation of the BW:FccR-f transfectants
This assay was described elsewhere [28]. Briefly, in a standard assay, target cells were incubated with dilutions of human sera, IVIG, the anti-hCD20 IgG isotype collection or trastuzumab in D-MEM with 10% (vol/vol) FCS for 30 min at 37uC. Cells were washed before co-cultivation with BW:FccR-f transfectants (ratio E:T 20:1) for 16 h at 37uC in a 5% CO 2 atmosphere. Then mIL-2 secreted was measured by ELISA. When applied to VACV infected cells, a previous step of UV-inactivation at 4000 Jules/m 2 and 2 steps of washing with PBS were performed before opsonization with Abs. When applied to the inhibition through soluble vFccR, soluble proteins were added concomitantly with BW:FccR-f transfectants. For herpesviral late antigens IgGdependent activation of BW:FccRIIIA cells, late phase gene expression was blocked by the use of phosphonoacetic acid (PAA) (250 mg/ml), which blocks viral genome replication and late gene expression. Afterwards, a co-cultivation assay was performed as described above.

Expression and purification of soluble V5-His tagged HCMV vFccR ectodomain proteins
The N-terminus of gp34 was amplified by PCR using the following primers 59-GCTTAGGGATCCATGCAGACCTA-CAGCACCCC-39) [22] and 59-TCTCACTAGTGGACCAC-TGGCGTTTTAAATC-39. Cloning of the N-terminus of gp68 was previously described [24]. The N-terminus of ICOSL (ICOS ligand) was amplified by PCR using the following primers 59-GAGGTAAGATCTCGCACCATGCGGCTGGGC-39 and 59-CTCTCACTAGTCGTGGCCGCGTTTTTC-39. Sequencing of the coding sequences showed an amino acid exchange in ICOSL from V 128 to I 128 but with no detectable functional difference. Each PCR product was cloned in pGene/V5-His B vector (Invitrogen, USA) in frame with the V5-His epitope tag using the restriction sites BglII and SpeI (italics in primer sequences) and then subcloned in pIRES-EGFP vector (Clontech, USA). For enhanced expression of gp34V5-His and gp68V5-His, c-Globin cloned from pSG5 vector (Stratagene, USA) was inserted into pIRES-EGFP between the CMV-IE promoter and the coding sequences. The plasmids were transfected in HEK293 cells using Superfect (Qiagen, Germany) and transfected cells were selected with 1.25 mg/ml of Geneticin (Sigma-Aldrich, Germany). After 4-5 days, supernatants were collected, volume reduced, diluted with PBS (1:3), adjusted to a 10 mM Imidazole concentration and passed over a His-Trap FF crude column (GE Healthcare, USA). Proteins were eluted in Imidazole/Phosphate buffer (250 mM Imidazole, 20 mM sodium phosphate, 500 mM NaCl) and then dialyzed to PBS. Comparable protein amounts were adjusted based in Western blot analysis using a-V5 antibody (Invitrogen, USA).

FACS analysis for HCMV and HSV surface antigen expression on infected cells
MRC-5 cells were infected with 1 PFU/cell of wt HCMV and HCMV DvFccR mutants for 72 h and with 10 PFU/cell of HSV-1 wt and DgE for 24 h. Cells were resuspended in PBS containing 2 mM EDTA, washed twice in PBS supplemented with 3% (vol/ vol) FCS. HCMV infected cells were stained with the F(ab) 2 preparation of Cytotect, goat anti-human-F(ab) 2 -Biotin, and Streptavidin-PE (AdB Serotec, UK) or Fcc fragment-FITC (Rockland Immunochemicals, USA). The comparability of infection of the different HCMV DvFccR mutants was controlled by intracellular staining of CMV nuclear antigens with CCH2 and DDG9 antibodies (Dako, Denmark) and goat anti-mouse-APC (BD Pharmingen, USA) after fixation with 1,5% PFA and permeabilization with PBS supplemented with 3% (vol/vol) FCS and 0,05% (vol/vol) Saponin. HSV infected cells were stained with the F(ab) 2 preparation of Cytotect, goat anti-human-F(ab) 2 -Biotin (AdB Serotec, UK) or Fcc fragment-Biotin (Rockland Immunochemicals, USA) and Streptavidin-APC (Jackson Immunoresearch, USA). After DAPI staining, 1-2610 4 living cells were obtained in a FACSCanto II using the FACS Diva software and analyzed with FlowJo (Tree Star Inc, USA).

CD107a NK cell degranulation assay
PBMCs were prepared from EDTA-blood of healthy donors using Lymphoprep (Axis-Shield, Norway) differential centrifugation. PBMCs were incubated during 3 h at 37uC to allow adherence of unwanted cells. Suspension cells were collected and resuspended in media containing 100 IU/ml of human rIL-2 (PromoKine, Germany) and incubated overnight at 37uC. Cells were resuspended and further processed to obtained polyclonal NK cells using a MACS negative selection NK cell isolation kit (Miltenyi Biotec, Germany). NK cell purity was tested in FACS and was usually above 96% (data not shown). For measuring degranulation by co-cultivation of immune IgG and a viral target, HCMV or HSV infected fibroblasts were opsonized with a serum of a healthy donor positive for HCMV and HSV or with IVIG. As a control, a healthy seronegative donor was also analyzed. Opsonization was done at 37uC for 30 min at 5% CO 2 . Two steps of washing with D-MEM 10% (vol/vol) FCS followed to remove unbound IgG. 1610 5 polyclonal NK cells (E:T ratio of 10:1) were added in each well and the CD107a assay was performed as elsewhere described [43]. Briefly, polyclonal human NK cells were incubated 4 h at 37uC in the presence of 6 mg/ml Golgi Stop (Monensin, BD Pharmingen, Belgium), 10 mg/ml Golgi Plug (BrefeldinA, BD Pharmingen, Belgium), and CD107-PeCy5 mAb (BD Pharmigen, Belgium). NK cells were collected, washed twice in ice cold PBS containing 2 mM EDTA and stained for extracellular markers (CD56, CD3). 1610 4 cells were counted and analyzed.  Figure S4 Ectopic expression of HSV-1 gE, HCMV gp68 and HCMV gp34 inhibit IgG1 mediated activation of FccRIIA. CD20 transfected 293T cells were infected for 16 hours with 2 PFU/cell of VACV wt or rVACV expressing gE (A) or gp68 and gp34 (B). After opsonization with 4 mg of rituximab (anti-hCD20 IgG1) and washing for removing unbound antibody, cells were co-cultivated with 1610 5 BW:FccRIIA-f 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 two independent experiments. Significance of results (Student's t-test) are presented in Table S1