Human cytomegalovirus antagonizes activation of Fcγ receptors by distinct and synergizing modes of IgG manipulation

Human cytomegalovirus (HCMV) is endowed with multiple highly sophisticated immune evasion strategies. This includes the evasion from antibody mediated immune control by counteracting host Fc-gamma receptor (FcγR) mediated immune control mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). We have previously shown that HCMV avoids FcγR activation by concomitant expression of the viral Fc-gamma-binding glycoproteins (vFcγRs) gp34 and gp68. We now show that gp34 and gp68 bind IgG simultaneously at topologically different Fcγ sites and achieve efficient antagonization of host FcγR activation by distinct but synergizing mechanisms. While gp34 enhances immune complex internalization, gp68 acts as inhibitor of host FcγR binding to immune complexes. In doing so, gp68 induces Fcγ accessibility to gp34 and simultaneously limits host FcγR recognition. The synergy of gp34 and gp68 is compelled by the interfering influence of excessive non-immune IgG ligands and highlights conformational changes within the IgG globular chains critical for antibody effector function.


Introduction
Human cytomegalovirus (HCMV) constitutes the prototypical human pathogenic b-herpesvirus found worldwide with high immunoglobulin G (IgG) sero-prevalence rates of 56-94% depending on the respective countries (Zuhair et al., 2019). A hallmark of cytomegalovirus infection is the establishment of a lifelong persistence with recurring phases of latency and reactivation of productive infection and horizontal spread in presence of adaptive immune responses. HCMV encodes the largest known transcriptome of human viruses (Stern-Ginossar et al., 2012), giving rise to an equally large antigenic proteome including a huge and varied arsenal of immunoevasins (Berry et al., 2020;Hengel et al., 1998) that counteract immune recognition of infected cells facilitating virus persistence, shedding, and superinfection of sero-positive hosts. While primary HCMV infection of healthy individuals usually remains undetected, it can cause severe symptoms in the immunocompromised. Besides antiviral therapy involving nucleotide analogs, concentrated HCMV-immune IgG preparations (e.g. Cytotect) are used to prevent infection of immunocompromised patients including the prevention of congenital infection in primary infected pregnant women with varying degrees of success (Kagan et al., 2019;Revello et al., 2014). Generally, HCMV is observed to withstand a humoral immunity even replicating and disseminating in the presence of highly neutralizing immune sera in vitro and clinical isolates of HCMV tend to disseminate via cell-to-cell spread, thus avoiding an encounter with neutralizing antibodies (Falk et al., 2018). This, however, cannot fully explain the resistance of HCMV to a humoral response leading to virus dissemination across several organs. Notably, HCMV encodes a set of Fcg-binding glycoproteins (viral FcgRs, vFcgRs) that have been shown to antagonize host FcgR activation by immune IgG (Corrales-Aguilar et al., 2014b). In this previous study, we showed that HCMV gp34, gp68 and HSV-1 gE/gI efficiently antagonize the activation of human FcgRs. By attacking the conserved Fc part of IgG, HSV-1 and HCMV are able to counteract potent antiviral immune responses including antibody-dependent cellular cytotoxicity (ADCC). As it has become increasingly evident that Fcg mediated immune control is essential not only for the effectiveness of non-neutralizing but also neutralizing IgG antibodies (DiLillo et al., 2014;Forthal et al., 2013;Horwitz et al., 2017;Van den Hoecke et al., 2017), a mechanistic and causal analysis of this evasion process is highly warranted in the pursuit of better antibody based intervention strategies targeting herpesviruses and HCMV in particular. While several herpesviruses encode vFcgRs, HCMV is the only virus known so far that encodes more than one individual molecule with the capacity to bind Fcg. Specifically, HCMV encodes four distinct molecules which share this ability: gp68 (UL119-118), gp34 (RL11), gp95 (RL12), and gpRL13 (RL13) (Atalay et al., 2002;Corrales-Aguilar et al., 2014a;Cortese et al., 2012;Sprague et al., 2008). Turning to other species, mouse CMV (MCMV) encoded m138 has been shown to bind Fcg, yet it has more prominently been associated with a variety of unrelated functions proving m138 not to be a strict homolog of the HCMV counterparts (Arapovic´et al., 2009;Lenac et al., 2006). We recently found Rhesus CMV (RhCMV) to encode an Fcg-binding protein in the Rh05 gene (RL11 gene family) seemingly more closely related to its HCMV analog (Kolb et al., 2019). This is supported by the fact that gpRh05, as HCMV vFcgRs gp34 and gp68, is able to generically antagonize activation of all macaque FcgRs. While it is clear that by targeting the invariant part of the key molecule of the humoral immune response, vFcgRs have the potential to manipulate a multitude of antibody mediated immune functions, their role in vivo has yet to be determined. While the function of HCMV vFcgRs gp34 and gp68 as antagonists of host FcgRs has been established (Corrales-Aguilar et al., 2014b), the underlying mechanism(s) had not been addressed yet. In recent years it has been shown that gp68 and gp34 are able to engage in antibody bipolar bridging (ABB) forming ternary complexes consisting of antigen, antibody, and vFcgR (Corrales-Aguilar et al., 2014a;Corrales-Aguilar et al., 2014b;Sprague et al., 2008). Moreover, gp68 has been shown to bind IgG in a 2:1 ratio and has the ability to internalize and translocate IgG to lysosomal compartments, while gp34 has been shown to form predominantly homo-dimeric structures (Ndjamen et al., 2016;Sprague et al., 2008). However, no eLife digest Human cytomegalovirus is a type of herpes virus that rarely causes symptoms in healthy people but can cause serious complications in unborn babies and in people with compromised immune systems, such as transplant recipients.
The virus has found ways to successfully evade the immune system, and once infected, the body retains the virus for life. It deploys an arsenal of proteins that bind to antibodies, specialized proteins the immune system uses to flag virus-infected cells for destruction. This prevents certain cells of the immune system, the natural killer cells, from recognizing and destroying virus-infected cells.
These immune-evading proteins are called viral Fc-gamma receptors, or vFcgRs. While it has been previously shown that these receptors are able to evade the immune system, it remained unknown how exactly they prevent natural killer cells from recognizing infected cells. Now, Kolb et al. show that the cytomegalovirus deploys two vFcgRs called gp34 and gp68, which work together to block natural killer cells. The latter reduces the ability of natural killer cells to bind to antibodies on cytomegalovirus-infected cells. This paves the way for gp34 to pull virus proteins from the surface of the infected cell, making them inaccessible to the immune system. Neither protein fully protects virus-infected cells on its own, but together they are highly effective.
The experiments reveal further details about how cytomegalovirus uses two defense mechanisms simultaneously to outmaneuver the immune system. Understanding this two-part viral evasion system may help scientists to develop vaccines or new treatments that can protect vulnerable people from diseases caused by the cytomegalovirus.
studies have yet been performed in the context of HCMV infection investigating the coincident disposition of gp34 and gp68 at the plasma membrane and their functional interaction during the early and late phase of HCMV replication. Here, we show gp34 and gp68 to antagonize host FcgR activation by distinct but highly cooperative modes of Fcg targeting, leading to efficient evasion from antibody mediated immune control by division of labor.

Results
gp34 and gp68 simultaneously bind to distinct regions on IgG. gp68 binding to IgG has been mapped to the CH2-CH3 interdomain region of Fcg (Sprague et al., 2008). Accordingly, in a first experiment we set out to narrow down the contact site of gp34 on IgG utilizing a methodology previously used to characterize HSV-1 gE and HCMV gp68 (Sprague et al., 2004;Sprague et al., 2008). To this end we infected CV-1 cells with recombinant vaccinia viruses (rVACV) encoding either human FcgRIIA, FcgRI or HCMV vFcgRs gp34 and gp68 (Sprague et al., 2008). After metabolic [ 35 S]-Met/Cys labeling, Fcg-binding proteins were precipitated from cell lysates using CNBr-Sepharose coupled with human IgG1-Fc in its wild-type form (wtFc) or as a mutated variant (nbFc) with a scrambled CH2-CH3 interdomain amino acid sequence designed and provided by P. Bjorkman (Caltech, California, USA) (Sprague et al., 2004;Sprague et al., 2008). Expectedly, gp68 was only able to bind wtFc but not nbFc whereas gp34, comparable to human FcgRI, retained binding to both wtFc and nbFc ( Figure 1A). While the high affinity FcgRI does not require the CH2-CH3 region to bind to the lower hinge of IgG, FcgRIIA and FcgRIII show lower affinity to monomeric IgG and utilize additional distinct residues for binding to Fcg, which include the CH2 region adjacent to the lower hinge region of IgG (Shields et al., 2001;Sprague et al., 2008;Wines et al., 2000). Consequently, FcgRII exhibited reduced binding to nbFc ( Figure 1A). Since gp34 showed intact binding similar to host FcgRI but not FcgRIIA we surmised that gp34 binding involves the hinge region of monomeric IgG. To validate this assumption further gp34 binding to a variant form of hIgG1 with point mutations in the lower hinge region (Leu234Ala and Leu235Ala, named LALA) exhibiting deficient interaction with human FcgRs (Hessell et al., 2007) was analyzed ( Figure 1B). Cell lysates were incubated with either B12 wild-type antibody recognizing human immunodeficiency virus (HIV)-encoded gp120 or a B12-LALA variant. PGS-precipitation of FcgRI/CD64, exclusively recognizing the hinge region on IgG, was almost abrogated by the mutations in the LALA variant. Similarly, gp34 was sensitive to the mutations, albeit to a lower extent than FcgRI/CD64. As expected, the interaction of gp68 with B12 was unaffected by the LALA point mutations. Next, as gp34 and gp68 recognize non-overlapping regions on IgG we set out to test, if both molecules are able to simultaneously associate with IgG. To this end, we generated C-terminal His-or strep-tagged variants of gp34 and gp68 that lack their respective transmembrane and cytosolic domains (soluble gp34 and gp68, or sgp34 and sgp68). These proteins were produced by transfected 293 T cells and secreted into the cell culture supernatant (Figure 1-figure supplement 1). Supernatants of sgp34 and sgp68 producing cells were mixed with humanized anti-hCD20 IgG1 (Rituximab, or Rtx) before being precipitated via Ni 2+ -NTA-Sepharose beads. A non-Fcg-binding point mutant of sgp34 (sgp34mtrp, W65F mutation) was identified and used as a control molecule (Figure 1-figure supplement 2). When precipitating Histagged sgp68 in the presence of monomeric IgG, we observed co-precipitation of sgp34 but no coprecipitation of sgp34mtrp ( Figure 1C). To further substantiate this observation, we designed an ELISA based setup using the same supernatants as above and performed the assay as depicted schematically in Figure 1D. In brief, titrated amounts of strep-tagged sgp34 or sgp34mtrp were immobilized on a 96-well adsorption plate (1˚vFcgR) via pre-coated biotin. 1˚vFcgRs were then incubated with either Rtx (IgG1) or a Rtx IgA isotype and incubated with His6-tagged sgp68 followed by detection with an anti-His-HRP antibody. Here, we observed dose-dependent binding of sgp68 to titrated amounts of immobilized sgp34. sgp68 could only be detected in the presence of IgG1 but not IgA. Furthermore, sgp68 binding in the presence of IgG1 and sgp34mtrp in this setup does not exceed background levels. Taken together, we conclude that gp34 and gp68 are able to simultaneously bind to IgG with gp34 binding to the upper hinge region and gp68 binding to the CH2-CH3 interface domain on IgG.
gp34 and gp68 exhibit synergistic antagonization of FcgR activation. gp34 (RL11) and gp68 (UL119-118) have previously been shown to antagonize FcgR activation independently (Corrales-Aguilar et al., 2014b). As gp34 and gp68 appear to have a redundant inhibitory function and are . gp34 and gp68 simultaneously bind to IgG using distinct epitopes. (A) CV-1 cells were infected with rVACVs expressing gp34, gp68, or the host Fc-receptors FcgRIIA and FcgRI at a multiplicity of infection of 4 for 14 hr before metabolic labeling. Proteins were precipitated using either wtFc or nbFc coupled with CNBr-activated Sepharose. Dissociated immune complexes were separated by 10% SDS-PAGE. IP, immunoprecipitation. Shown is one out of two independent experiments. (B) CV-I cells were infected and metabolically labeled as above. Lysates were incubated with B12 or B12- Figure 1 continued on next page able to simultaneously bind to IgG we utilized a previously established FcgR activation assay (Corrales-Aguilar et al., 2013) to assess their ability to antagonize FcgR activation individually or in combination. In brief, MRC-5 or human foreskin fibroblasts (HFF) were infected with HCMV AD169-pBAC2-derived mutant viruses lacking different combinations of vFcgRs. The other vFcgRs gp95 (RL12) and gpRL13 (RL13) were deliberately excluded in this study. RL13 (Cortese et al., 2012), a potent inhibitor of HCMV replication , is notoriously mutated or absent from the HCMV genome in cell culture passages  but has not yet been described to antagonize FcR activation. RL12 is one of the most polymorphic genes of HCMV (Dolan et al., 2004) with only minor surface expression in the context of AD169 infection (Corrales-Aguilar et al., 2014b). Due to remarkably low conservation of RL12 across HCMV strains, we focused on the wellconserved and previously described vFcgRs gp34 and gp68. Infected cells were incubated with titrated amounts of a HCMV-IgG hyperimmunoglobulin preparation (Cytotect) and co-cultured with . This showed no statistically significant effect for singular vFcgRs, but a strong and significant antagonization by both vFcgRs in combination. To further confirm this key finding we measured degranulation of primary NK cells in response to vFcgR expression to assess antagonization of ADCC. Peripheral Blood Mononuclear Cells (PBMCs) from three donors were incubated with HCMV-infected opsonized human fibroblasts generated as above and CD107a positivity of NK cells was measured after 6 hr of co-culture. In line with the reporter cell assay, primary NK cell activation in response to target cells decorated with anti-HCMV antibodies was antagonized by a virus expressing both gp34 and gp68 with high significance, while the mutant viruses lacking gp34 or gp68 showed drastically lower antagonistic potential ( Figure 2B). From these observations we conclude that gp34 and gp68 antagonize FcgRs by synergizing modes of action, suggesting cooperation, at least in the absence of gp95 (RL12) and gpRL13 (RL13).

HCMV gp34 enhances internalization of immune complexes
As we observed gp34 and gp68 to work in concert to maximize FcgR inhibition, we set out to elucidate the mechanisms by which the vFcgRs achieve this synergistic effect. When comparing the amino acid sequences of the cytosolic domains of gp34 and gp68 it is revealed that both molecules encode distinct sorting motifs ( Figure 3A; Atalay et al., 2002) (source: AD169 pBAC-2 sequence). The cytosolic tail of gp68 harbors an YXXF motif seven aa downstream of the transmembrane domain. Such a motif has been described as a marker for lysosomal targeting (Bonifacino and Traub, 2003) and is LALA and IgG was precipitated using PGS. All samples were de-glycosylated using EndoH resulting in double bands for gp68 (Sprague et al., 2008). Shown is one out of two independent experiments. (C) Soluble vFcgRs were tested for simultaneous IgG1 (Rtx) binding by Ni-NTA-Sepharose coprecipitation and subsequent immunoblot in the presence of IgG (Rtx). gp34 and gp34mtrp were streptavidin-tagged; gp68 was 6xHis-tagged. All samples were deglycosylated using PNGaseF resulting in double bands for gp68 (Sprague et al., 2008). LC = loading control, P = precipitate. Shown is one out of two independent experiments. (D) Soluble vFcgRs as in B were tested for simultaneous IgG1 (Rtx) binding via ELISA as schematically depicted. An anti-CD20 IgA molecule served as a negative control. The 1˚sgp34-strep or sgp34mtrp layers were generated by incubation of titrated amounts of supernatants from soluble vFcgR producing cells on coated biotin. Supernatants were diluted in PBS. 2˚sgp68-His detection was performed using 1:2 diluted supernatant accordingly. Graph shows averages from two independent experiments performed in technical replicates (Figure 1source data 1). Error bars = SD. Two-way ANOVA.
The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Relates to Figure 1D bar graph. in line with the lysosomal translocation of gp68 shown in a previous study (Sprague et al., 2008). In HCMV gB, a non-Fcg-binding HCMV encoded envelope glycoprotein, the location of two such motifs is further away from the transmembrane domain indicating it being destined for general endocytosis rather than immediate degradation, which is shared by another HCMV vFcgR, gpRL13, found primarily in intracellular compartments as well as HSV-1 gE, forming a heterodimeric gE/gI vFcgR described to internalize IgG and recycle back to the cell surface ( Figure 3-figure supplement 1; Bonifacino and Traub, 2003;Cortese et al., 2012;Ndjamen et al., 2016;Sprague et al., 2004). Conversely, the cytosolic tail of gp34 encodes a di-leucine [D/E]XXX[LL/LI] motif which we find to be unique among surface-resident vFcgRs. Therefore we suspected gp34 and gp68 to possess different dynamics regarding IC internalization. As internalization studies in the context of HCMV infection proved not to be feasible due to lacking gp34-and gp68-specific antibodies for tracing, we aimed to establish a gain-of-function cell-based experimental model. To this end, we characterized the surface dynamics of vFcgR expressing transfected 293 T cells stably expressing a model antigen (hCD20) recognized by Rituximab (RTX). In order to manipulate internalization, we chose to replace the transmembrane and cytosolic domains of gp34 and gp68 with the according sequences from human CD4 (hCD4) as it contains a non-canonical di-leucine-based sorting motif (SQIKRLL) in its C-terminal cytoplasmic tail (CD4-tailed). To be recognized by AP-2 the serine residue upstream of the di-leucine motif in hCD4 needs to be phosphorylated in order to mimic the negatively charged glutamate or aspartate residues of a classical di-leucine motif (Pitcher et al., 1999). In a first experiment we ensured equal expression of the transfected constructs utilizing the polycistronic expression of GFP from a pIRES_eGFP expression vector to gate on transfected cells ( Figure 3B). GFP-positive cells were also detected for Fcg binding by a surface stain using a PE-Tex-asRed conjugated human Fcg fragment ( Figure 3C). This revealed that upon recombinant expression, surface exposition of Fcg binding gp68 is drastically increased when CD4-tailed although overall protein expression was comparable and all constructs bear original signal peptides ( Figure 3B). This can be attributed to the abrogation of its internalization evidenced by the fact that internalization of complete IC was drastically reduced with CD4-tailed gp68 when tracking hCD20/ Rtx ICs using pulse-chase flow cytometry ( Figure 3D). Conversely, while gp34 showed only mild differences in surface exposition when altered in the same way ( Figure 3C), it proved to rapidly internalize complete Rtx-CD20 IC only with its native cytosolic tail intact ( Figure 3D). Given that both molecules spontaneously internalize non-immune IgG (Figure 3-figure supplement 2), this could hint at different routes of trafficking following internalization. Specifically, as it has been shown that gp68 internalizes IgG-Fc translocating to the lysosomal compartment for degradation (Ndjamen et al., 2016), the difference seen for gp34 here suggests a recycling route as described for HSV-1 gE/gI (Ndjamen et al., 2014). Further, while we also observe gp34 to more efficiently internalize even non-immune IgG, the still rapid internalization of non-immune IgG by gp68 seems not to translate to the internalization of IC regarding efficiency (Figure 3-figure supplement 2), again in line with a previously published observation (Ndjamen et al., 2016). As we observed significantly more efficient internalization of IC by gp34wt compared to gp68wt or HSV-1 gEwt ( Figure 3D, Figure 3-figure supplement 2), we conclude that a major task of gp34 compared to gp68 is mediating the efficient internalization of IC, a feature likely linked to its particular di-leucine cytosolic motif.  FcgRIII binding to opsonized cells is reduced in the presence of gp68 but not gp34 As previously reported (Atalay et al., 2002;Corrales-Aguilar et al., 2014b) and confirmed in this study, we find that in the context of HCMV infection gp68 is more surface resident compared to gp34 ( Figure 6A). Therefore, we next set out to test if the antagonizing effect of membrane-resident gp68 on FcgR activation can be attributed to a block of host FcgR binding to cell surface IC rather than IC internalization. To test this hypothesis, we established a flow cytometry based assay using vFcgR transfected 293 T-CD20 cells opsonized with Rtx and assayed for FcgR binding using soluble His-tagged ectodomains of human FcgRs, which are sequence identical between FcgRs IIIA and IIIB ( Figure 4B). In this assay we compared gp34 and gp68 regarding their ability to interfere with FcgR binding to cell surface IC. Advantageously, CD4-tailed vFcgRs, besides circumventing confounding effects of internalization on our binding assay, closely mimic the relative surface density of gp34 and gp68 found on the plasma membrane of an HCMV-infected cell judged by human Fcg binding ( Figure 3C, Figure 5A; Corrales-Aguilar et al., 2014b). To ensure equal density of antigen upon recombinant vFcgR expression, CD20 levels were directly measured and found only in the case of CD99 expression to be slightly reduced, which served as a non-Fcg-binding control molecule ( Figure 4A). We found gp68 but not gp34 to significantly reduce binding of FcgRIIIA to cell surface immune complexes compared to CD99 ( Figure 4C). This finding can be explained with certain CH2-CH3 region residues of IgG playing a subordinate, yet significant role in FcgRIII binding to IgG (Shields et al., 2001). Additionally, we found the effect of gp68 to be dependent on the accessibility of the CH2-CH3 interface region on IgG as pre-incubation with Protein G prevented the inhibition by gp68 and fully restored FcgRIIIA binding to Rtx ( Figure 4C). These observations further narrow down the binding region of gp68 to involve the above-mentioned residues as opposed to Protein G which has been shown to not interact with these residues (Sauer-Eriksson et al., 1995). Finally, this conclusion is further substantiated by our observation that FcgRI, which binds to IgG independently of the above-mentioned residues in the CH2-CH3 interdomain region (Shields et al., 2001), is not blocked by gp68 or gp34 (Figure 4-figure supplement 1).

Human FcgR binding to opsonized HCMV-infected cells is reduced by viral gp68
In order to test if our previous findings regarding the block of human FcgRIII binding to immune complexes in the presence of gp68 translate into a loss-of-function approach, MRC-5 cells were infected with AD169/pBAC2-derived HCMV mutants as described above. Uniformity of HCMV antigen expression and differential surface Fcg binding indicating vFcgR expression between the different virus mutants was assured using a F(ab') 2 preparation of purified human IgG containing HCMVspecific IgG, Cytotect, or a conjugated human Fcg fragment (human Fcg-Texas Red, TR), respectively ( Figure 5A). FcgR binding to opsonized-infected cells was detected by flow cytometry as described above and schematically depicted in Figure 5B. Expectedly, as we observed stronger Fcg binding to cells infected with gp68 proficient viruses compared to a gp34 expressing virus ( Figure 5A), we also found more total IgG bound to the surface of cells infected with a gp68 expressing HCMV virus compared to a gp34 expressing virus ( Figure 5C, upper panel). Along these lines, we find FcgRIII to bind Internalization of CD20/Rtx immune complexes in dependence of vFcgR expression was measured by loss of surface signal over time in a pulse-chase approach detecting residual surface complexes via a PE-conjugated mouse-anti-human-IgG antibody. 293 T cells stably expressing CD20 were transfected with the indicated constructs. HSV-1 gE was co-transfected with gI to form a functional heterodimer (Ndjamen et al., 2014). Left: Exemplary experiment comparing internalization rates between native vFcgRs (wt) and their respective CD4-tailed variants. Right: Internalization rates 1 hr post pulse. Two independent experiments (Figure 3-source data 1). Error bars = SD. One-way ANOVA.
The online version of this article includes the following source data and figure supplement(s) for figure 3: Source data 1. Relates to Figure 3D bar graph.  Source data 1. Relates to Figure 5D bar graph.  gp68-bound non-immune IgG ( Figure 5-figure supplement 1), albeit at a markedly lower rate compared to immune IgG (Figure 4) in line with FcgRs naturally showing higher affinity toward antigen-bound IgG (Bruhns et al., 2009). As the Cytotect formulation contains an abundance of nonimmune IgG, binding of host FcgR ectodomains as shown exemplarily in Figure 5C (lower panel) was normalized to levels of total surface bound IgG within each experiment ( Figure 5C, upper panel). Evaluating the results from three independent experiments performed with independent virus preparations confirmed a strong relative reduction in FcgRIIIA binding to opsonized HCMVinfected cells in the presence of gp68, but not gp34 ( Figure 5D). This finding is in line with our previous gain-of-function experiments ( Figure 4C) again showing an approximate 60% reduction in FcgRIII binding. While not further elaborated on in this study, we also measured binding of FcgRs I, IIA, and IIB/C in the same setup and observed that binding of FcgRI was only slightly reduced in the presence of either vFcgR, but clearly reduced in the presence of both molecules. Conversely, gp68 showed a similar effect on FcgRs IIA and IIB/C as observed for FcgRIII ( Figure 5-figure supplement  2). This indicates a comparable antagonistic mechanism of gp68 on FcgRs IIA and IIB/C but shows again FcgRI to be more resistant to gp68.

Cooperative antagonization of FcgR activation is achieved by combining gp68-mediated block of FcgR binding and subsequent gp34 internalization
While we delineated distinct mechanisms by which gp34 or gp68 are able to counteract FcgRII/III activation, we did not observe efficient antagonization of FcgR function by gp34 or gp68 individually in the context of HCMV deletion mutant infection ( Figure 2). Therefore, we next wanted to test the antagonistic potential of gp68 or gp34 individually in the absence of non-immune IgG and in a gainof-function setting. To this end, we conducted an FcgR activation assay with Hela cells co-transfected with Her2 as a model antigen and the indicated vFcgRs followed by incubation with titrated amounts of anti-Her2 IgG1 mAb (herceptin, Hc) ( Figure 6A). Using this approach, gp68 or gp34 individually confirmed to antagonize FcgRIII activation. However, the more membrane resident gp68-CD4 showed a markedly stronger antagonistic effect compared to gp68wt, indicating a more prominent membrane-residence of gp68 to be beneficial regarding evasion from FcgR recognition despite its lower capacity to internalize ICs ( Figure 3D). Conversely, when comparing unaltered gp34 to its less internalized gp34-CD4 variant we observed an opposite effect indicating internalization to be a major condition of gp34 driven antagonization of FcgR activation. Next, in an approach mimicking HCMV immune sera we tested if the addition of non-antigen-specific IgG impairs the antagonization of FcgR activation by gp34 or gp68. To this end, we added 10 mg/ml TNFa-specific Infliximab (Ifx) IgG1 given concomitantly with the reporter cells and graded amounts of Hc IgG1 ( Figure 6B). This showed that indeed the presence of an excess of non-immune IgG interferes with both gp68 and gp34 antagonization in a dose-dependent manner. As ultrapure monomeric IgG does not activate the reporter cells on its own, shown by the samples that were treated only with Ifx but not Hc, this effect can be attributed to displacement of immune IgG from the vFcgRs resulting in restoration of FcgRIII activation. Synergism between gp34 and gp68 was optimal at a 100:1 ratio of non-immune Ifx over Hc ( Figure 6B, right panel) indicating that vFcgRs are particularly adapted to work in the presence of excess non-immune IgG, supporting our previous observation with human hyperimmunoglobulin Cytotect (see Figure 2). On the other hand, host FcgRs have been shown to bind IgG immune complexes with a higher affinity compared to monomeric IgG (Bruhns et al., 2009), limiting the efficacy of monomeric IgG in attenuating FcgR activation. Finally, we addressed cooperative antagonization by gp34 and gp68 in a loss-of-function approach in the context of HCMV infection. When using the humanized anti-HCMV mAb MSL-109 targeting HCMV gH, we found that low gH levels expressed on the surface of HCMV-AD169-infected cell are insufficient to detect FcgR triggering ( Figure 6-figure supplement 1). To overcome this experimental problem, we generated Her2expressing BJ fibroblasts exhibiting high levels of Her2 on the cell surface. Infected Her2 BJ fibroblasts were opsonized with titrated amounts of Herceptin before cells were analyzed by FcgRIIIA reporter cells ( Figure 6C). Although not statistically significant, this approach clearly demonstrated that compared to our observations with hyperimmunoglobulin Cytotect (see Figure 2), natively expressed gp34 as well as gp68 are able to antagonize FcgRIIIA activation individually. When both vFcgRs were expressed by HCMV-infected cells, a markedly stronger antagonistic effect was seen (p=0.0252).  Following the reveal of synergistic modes of action, we next explored cooperativity between gp34 and gp68 in a setup of reduced complexity. Specifically, in order to elaborate on the cooperativity of gp34 and gp68 regarding the here highlighted main mechanisms of internalization (gp34) and host FcgR-binding blockade (gp68) we chose to co-express gp34 and Her2 antigen while adding soluble gp68 (sgp68, Figure 1-figure supplement 1), or a soluble functional deficient gp34 point mutant as a control (sgp34mtrp, Figure 1-figure supplement 1 and Figure 1-figure supplement 2), together with the reporter cells after the removal of unbound Hc. Here we observe that the ectodomain of gp68 is sufficient to enhance the antagonistic effect of gp34 ( Figure 6C).
In summary, we conclude that (i) gp34 is designed for internalization of IC while gp68 blocks FcgR binding to IC; (ii) gp34 and gp68 are able to antagonize FcgR activation individually when faced with titrated amounts of immune IgG, but non-immune IgG interferes with this inhibition; (iii) gp34 and gp68 show cooperativity in attenuating FcgR activation particularly under conditions of high excess of non-immune IgG.

Discussion
Here we delineate for the first time synergistic modes of action involving two independent viral factors to efficiently achieve evasion from antibody-mediated immune cell surveillance (Figure 7). The novelty of these mechanisms acting cooperatively on the surface of an infected cell has major implications for our perception of how viruses have co-evolved multifactorial strategies to compete in a molecular arms race with the host at the junction between adaptive (humoral) and innate immunity. In particular, this knowledge refines our understanding of how HCMV can efficiently evade from a fully developed, broadly polyclonal and affinity-matured IgG response using its large coding capacity to ensure its reactivation, shedding, spread, and eventually transmission.
Recognizing the conserved Fc part of IgG and exhibiting a very similar FcgR antagonization profile in reductionistic gain-of-function approaches (Corrales-Aguilar et al., 2014b), the two membrane-resident glycoproteins gp34 (RL11) and gp68 (UL119-118) appeared at first glance merely redundant. This left the puzzling question as to why HCMV (and cytomegaloviruses in general) have developed a whole arsenal of antagonists, unlike other viral and microbial pathogens with only singular Fc-binding glycoproteins (Berry et al., 2020;Corrales-Aguilar et al., 2014a;Falugi et al., 2013). However, when carefully examining their effect in a loss-of-function approach based on opsonized cells infected with a complete set of targeted HCMV gene deletion mutants, we convinced ourselves that gp34 and gp68 actually depend on one another to efficiently antagonize NK cell degranulation (Figure 2). This is explained by a number of individual features of gp34 and gp68 that only in conjunction can amount to antagonistic efficiency. transfected as indicated in combination with Her2 antigen and incubated with titrated amounts of Herceptin (mg per 100 ml) followed by FcgRIII activation assessment. FcgR activation was assessed in the absence or presence of excess of non-immune IgG by addition of 1 mg per 100 ml Infliximab (Ifx). Hc, Ifx, and reporter cells were added concomitantly. Titrations show one exemplary experiment. Error bars = SD. Bar graphs show combined averages from three independent experiments performed in technical replicates at 0.01 mg Hc normalized to CD99 control ( Figure 6-source data 1). Error bars = SD. Two-way ANOVA. (C) gp34 and gp68 antagonize FcgRIIIA activation individually in the context of viral infection. BJ-Her2 cells were infected with the indicated human cytomegalovirus (HCMV) AD169 deletion mutants (MOI = 2). 72 hr post infection, cells were opsonized with titrated amounts of Herceptin and incubated with FcgRIIIA reporter cells. mIL-2 expression was quantified via anti-mIL-2 sandwich ELISA. Two independent experiments performed in technical replicates. Error bars = SD. Bar graph shows area under curve (AUC) comparison ( Figure 6-source data 2). Error bars = SD. One-way ANOVA. (D) gp68 cooperatively enhances antagonistic potential of gp34. Hela cells were co-transfected with gp34 and Her2 antigen and incubated with titrated amounts of Herceptin followed by FcgRIII activation assessment. gp34 expressing cells were supplemented with sgp68 or sgp34mtrp from soluble vFcgR producer cells at a 1:4 dilution. Soluble vFcgRs and reporter cells were incubated concomitantly after removal of pre-incubation with Hc to avoid saturation of sgp68 with unbound Hc. Titrations show one exemplary experiment. Error bars = SD. Bar graphs show combined averages from three independent experiments performed in technical replicates at 0.01 mg Hc normalized to CD99 control ( Figure 6source data 3). Error bars = SD. Unpaired t-test. The online version of this article includes the following source data and figure supplement(s) for figure 6: Source data 1. Relates to Figure 6B bar graph. Source data 2. Relates to Figure 6C bar graph. Source data 3. Relates to Figure 6D bar graph.

gp34 efficiently internalizes IC
While we find both, gp34 and gp68, able to internalize monomeric IgG as well as IC (Figure 3, Figure 3-figure supplement 2), gp34 consistently shows more rapid internalization compared to gp68 (Figure 3). Consequently exchange of the cytosolic domain of gp34 leads to strongly reduced internalization and subsequently to a reduction in FcgR antagonization ( Figure 6). Looking closely at the cytosolic domains of all identified cytomegalovirus vFcgRs reveals a unique [D/E]XXX[LL/LI] cytosolic motif present in gp34. Conversely, the HCMV FcgRs gp68, gp95, and gpRL13 all share only a cytosolic YXXF motif, as the recently identified RhCMV encoded vFcgR gpRH05 (RL11 gene family) and its homologs which are conserved in Old World monkey CMV species (Kolb et al., 2019). Further comparing the cytosolic sorting motifs between other membrane-resident glycoproteins of HCMV it seems that the YXXF motif of gp68 fulfills a more general purpose of limiting the overall surface exposure of concerned HCMV antigens (examples listed in Figure 3-figure supplement 1; Corrales-Aguilar et al., 2014b;Ndjamen et al., 2016). While the YXXF motif of gp68 lies within seven aa distance to the transmembrane domain, in line with it being translocated to lysosomal compartments (Bonifacino and Traub, 2003;Ndjamen et al., 2016), the YXXF motif is additionally linked to specific targeting functions. For example, HSV-1 gE has been shown to bind in a pHdependent manner not observed for any other HCMV encoded vFcgR (Sprague et al., 2004;Sprague et al., 2008) which fits to it being destined for recycling rather than degradation (Ndjamen et al., 2014). This is in line with its YXXF motif being more distant to its transmembrane domain within around seven amino acids (Bonifacino and Traub, 2003; Figure 3-figure supplement 1). Similar membrane-distal YXXF motifs also exist in the cytosolic domains of HCMV encoded gpRL13 and gB, but not the model antigen Her2 which we explored in Figure 6C, perhaps explaining the limited synergistic inhibition that was observed. These findings highlight the di-leucine motif HCMV gp68

HCMV gp34
FcγR binding block simultaneous binding internalization HCMV infected cell NK NK NK Figure 7. Graphical summary. NK cells elicit a powerful antibody-mediated antiviral response through antibody-dependent cellular cytotoxicity (ADCC). gp68 (ochroid) binds IgG in a 2:1 ratio reducing, but not abolishing accessibility of immune complexes to FcgRIII + (dark blue) immune effector cells such as NK cells. gp34 (red, natively forming a dimer [Sprague et al., 2008]) effectively internalizes immune complexes making them unavailable to surveilling FcgRIII + effector cells but cannot compete with FcgRIII for a similar binding region on IgG. Supported by functional evaluation, we propose a model in which g34 and gp68 work in cooperation to achieve efficient antagonization of antibody-mediated effector mechanisms.
found in gp34 to be unique among other surface-resident glycoproteins expressed by HCMV and all other vFcgRs known in CMV family members including RhCMV and MCMV (Kolb et al., 2019;Thäle et al., 1994). It remains to be explored as to what the further consequences of this seemingly unique internalization route is. However, as we also find gp34 to be incorporated into the virion (Reinhard, 2010) it is tempting to speculate that the potent internalization of IC via gp34 has an additional role besides evasion from antibody-mediated attack of an infected cell.

gp68 blocks binding of FcgRs II and III to IC
Although we find gp34 to efficiently internalize ICs from the cell surface, it does not manipulate host FcgR binding to ICs. This is remarkable given our data showing that gp34 binds to the hinge region of monomeric IgG, but implying that gp34 is not able to directly compete with host FcgRs for binding to IC on the plasma membrane when co-expressed with a target antigen. Conversely, gp68 binding to the CH2-CH3 interface domain also occupies a region on Fcg that includes residues involved in FcgRII and FcgRIII binding to Fcg (Shields et al., 2001;Sprague et al., 2008). This could explain the ability of gp68 to efficiently limit binding of FcgRs II and III to cell surface IC without directly competing for a shared binding region at the hinge region (Figure 1, Figures 4 and 5). The subordinate role of the above-mentioned residues on IgG regarding FcgRIII binding further ties into the observation that the blocking effect of gp68 is not total but more in line with the reported reduction of FcgRIII binding to IgG being approximately 40-70% when these residues are manipulated (Shields et al., 2001). Conversely, gp68 not showing a similar efficiency in blocking FcgRI binding to IC further supports this conclusion as FcgRI binding does not require the above mentioned CH2-CH3 region residues (Figure 3-figure supplement 1; Shields et al., 2001).

gp34 and gp68 cooperatively antagonize FcgR activation
Taken together, we conclude that neither gp68 nor gp34 individually are able to efficiently antagonize FcgR activation by IgG-opsonized viral antigens in the physiological context of HCMV infection (Figure 2), that is, in the presence of non-immune IgG mitigating their inhibitory potential ( Figure 6B and Figure 6C). Our data provide evidence that gp68 rather functions to increase IC accessibility to gp34 leading to efficient internalization of IC, presumably by a repeating process of gp34 recycling. This is supported by our observation that the ectodomain of gp68 is sufficient to increase the antagonistic efficiency of gp34 regarding FcgR activation ( Figure 6). Further, gp34 and gp68 are co-expressed at the cell surface throughout the protracted HCMV replication cycle and show simultaneous Fcg binding (Figure 1). Our findings imply that gp68 binding, as it does not use the same region as host FcgRs, evolved to ensure a consistent and evolutionary less vulnerable way of shifting Fcg accessibility in favor of gp34. The idea that gp68 has not evolved to directly compete with host receptors for IgG binding is also supported by the finding demonstrating gp68 to possess a lower affinity to Fcg when binding in a 2:1 ratio compared to host FcgRIIIA (K D1 :470 nM and K D2 :1.600 nM vs 700 nM) (Li et al., 2007;Sprague et al., 2008). The concept of gp68 driven accessibility shift also does not require gp68 to completely block FcgR binding to IC as subsequent gp34driven internalization is a continuous and fast process.

Beyond NK cells
In this study we mainly focused on the mechanisms by which gp34 and gp68 cooperate to antagonize CD16/FcgRIII as the primary receptor on NK cells associated with ADCC, one of the most powerful mechanisms of antibody-mediated virus control. However, it is already known that HCMV vFcgRs also antagonize activation of FcgRIIA (CD32) and FcgRI (CD64) (Corrales-Aguilar et al., 2014b) and we also consistently observed antagonization of the only inhibitory FcgR, FcgRIIB (unpublished observation). Similarly, we could recently show antagonization of all canonical Rhesus FcgRs I, IIA, IIB, and III by the RhCMV encoded vFcgR gpRH05 (RL11 gene family) (Kolb et al., 2019). Taken together with the data shown in this study that the underlying mechanism seems to be related between FcgRs IIA/B and III, we also speculate that more Fcg-binding host factors might be antagonized by similar mechanisms involving vFcgRs. In support of this view, besides gp34 and gp68, the HCMV RL11 gene family encodes additional vFcgRs. RL12 (gp95) and RL13 (gpRL13) likely are part of a larger arsenal of antibody targeting immunoevasins (Corrales-Aguilar et al., 2014a;Cortese et al., 2012). The fact that the interaction of gp34 and gp68 was analyzed in the absence of gp95 (RL12) and gpRL13 (RL13) is a major limitation of our study. Synergies between other or even all four vFcgRs are also conceivable and subject to further investigation. Altogether, our findings analyze the first mechanistic details of HCMV evasion from antibody-mediated control utilizing its vFcgR toolset. This deeper insight into the inner workings of such a process has consequences for the future evaluation and optimization of antibody-based treatment strategies targeting HCMV disease. In particular, our data will support the development of targeted intervention strategies that neutralize the function of gp34 and gp68, to increase the efficiency of IVIg treatment in the future.

Materials and methods
Key resources 2020) corresponding to AD169varL (Le et al., 2011) as parental genome. For the construction of the HCMV deletion mutants, a PCR fragment was generated using the plasmid pSLFRTKn (Atalay et al., 2002) as the template DNA. The PCR fragment containing a kanamycin resistance gene was inserted into the parental BAC by homologous recombination in E. coli. The inserted cassette replaces the target sequence which was defined by flanking sequences in the primers. This cassette is flanked by frt-sites which can be used to remove the kanamycin resistance gene by FLPmediated recombination. The removal of the cassette results in a single remaining frt-site. The deletion of multiple non-adjacent genes was conducted in consecutive steps. The gene TRL11 was deleted by use of the primers KL-DeltaTRL11-Kana1 (ACGACGAAGAG-GACGAGGACGACAACGTCTGATAAGGAAGGCGAGAACGTGTTTTGCACCCCAGTGAATTCGAGC TCGGTAC) and KL-DeltaTRL11-Kana2 (TGTATACGCCGTATGCCTGTACGTGAGATGGTGAGGTC TTCGGCAGGCGACACGCATCTTGACCATGATTACGCCAAGCTCC). The gene TRL12 was deleted by use of the primers KL-DeltaTRL12-Kana1 (CGGACGGACCTAGATACGGAACCTTTGTTG  TTGACGGTGGACGGGGATTTACAGTAAAAGCCAGTGAATTCGAGCTCGGTAC) and KL-Del-taTRL12-Kana2 (CCTTACAGAATGTTTTAGTTTATTGTTCAGCTTCATAAGATGTCTGCCCGGAAACG TAGCGACCATGATTACGCCAAGCTCC). The gene UL119 was deleted by use of the primers KL-DeltaUL119-Kana1 (TTGTTTATTTTGTTGGCAGGTTGGCGGGGGAGGAAAAGGGGTTGAACA-GAAAGGTAGGTGCCAGTGAATTCGAGCTCGGTAC) and KL-DeltaUL119-Kana2 (AGGTGACGC-GACCTCCTGCCACATATAGCTCGTCCACACGCCGTCTCGTCACACGGCAACGACCATGA TTACGCCAAGCTCC).
Ni 2+ -NTA Co-precipitation 10 ml soluble vFcgR supernatants were mixed 1:1 in the presence of 1 mg Rituximab. Antibody has to be given in a limiting amount to increase the probability of simultaneous binding to the same IgG molecule. Samples were incubated 1 hr at 4˚C and then mixed with freshly prepared Ni 2+ -NTA Sepharose beads (2 ml BV, cOmplete His-Tag purification resin) and incubated overnight at 4˚C in the presence of 20 mM Imidazole (rotate or shake). Beads were washed three times with PBS/20 mM Imidazole at 11,000 g and 4˚C. After the final wash, beads were either resuspended in sample buffer (Tris pH 6.8, SDS, Glycerol, 2-mercaproethanol, bromophenol blue) or subjected to a PNGase F digest before being supplemented with sample buffer (NEB, performed as suggested by the supplier). Samples were then denatured at 95˚C and analyzed via SDS-PAGE and subsequent immunoblot.

vFcgR-binding ELISA
We adapted a standard ELISA protocol to measure binding between soluble vFcgRs and target IgG. 96-well Nunc Maxisorp plates were coated with 1 mg of biotin in PBS. Plates were then blocked and incubated with titrated amounts of supernatants from soluble strep-tagged vFcgR producing cells (1v FcgRs). Supernatants were diluted in PBS. After incubation with 100 ng/well of target antibody, plates were incubated with a 2˚His-tagged soluble vFcgR (1:2 diluted) followed by detection using a HRP-conjugated anti-His antibody. Plates were measured using a Tecan Genios Pro microtiter plate reader at 450 nm/630 nm. Plates were washed three times between steps using PBS/0.05% Tw-20.

FcgR-binding assay
Transfected or infected vFcgR expressing cells were harvested using Accutase (Sigma-Aldrich) to retain surface molecules upon detachment. Harvested cells were washed in PBS, equilibrated in staining buffer (PBS, 3% FCS) and sedimented at 1000 g and 10˚C for 3 min. Cells were then incubated with staining buffer containing rituximab, Cytotect, or herceptin. Cells were then incubated in an adequate volume of staining buffer containing either mAbs, Fcg fragment, or FcgR ectodomains pre-incubated with an aHis-PE antibody (30 min, 4˚C). Human His-tagged FcgR ectodomains were used at a final concentration of 5 mg/ml (1:50 dilution from reconstituted 0.25 mg/ml stock solution; Sino Biological, 10389-H08H1). Pre-incubation with Protein G (Rockland, Biotin conjugated) was performed prior to incubation with FcgR ectodomains (diluted 1:100). Further incubation steps were carried out at 4˚C for 1 hr and followed by three washing steps in staining buffer. Dead cells were excluded via DAPI stain. Analysis was performed on a FACS Fortessa instrument (BD Bioscience).

IC internalization assay
Transfected 293 T-CD20 cells were harvested using Accutase (Sigma-Aldrich) to retain surface molecules upon detachment. Harvested cells were incubated with Rituximab (1 mg/well) for 1 hr at 4˚C in medium. Cells were then washed twice in medium containing 5% FCS and seeded into a 96-well plate at 2 Â 10 4 cells/well. Each reaction was performed on cells from one 96-well. Cells were then incubated at 37˚C in a 5% CO 2 atmosphere until being harvested at different time points ensuring regular re-suspension to avoid cell attachment over longer periods of time. After harvesting, cells were directly stained with ahuman-IgG-PE for 1 hr at 4˚C and fixed using 3% PFA. Analysis was performed on a FACS Fortessa instrument (BD Bioscience).

FcgR activation assay
The assay was performed as described earlier (Corrales-Aguilar et al., 2013). Briefly, target cells were incubated with titrated amounts of antibody (96-well format) in medium (DMEM) supplemented with 10% FCS for 30 min at 37˚C, 5% CO 2 . Cells were washed with medium (RPMI) and cocultured with BW5147-reporter cells (ratio E:T 20:1) expressing individual host FcyR ectodomains or CD99 as control for 16 hr at 37˚C in a 5% CO 2 atmosphere. Reporter cell mIL-2 secretion was quantified by subsequent anti IL-2 sandwich ELISA as described previously (Corrales-Aguilar et al., 2013).

NK cell degranulation assay
PBMCs were purified from healthy donor blood by centrifugation via Lymphoprep Medium according to the supplier instructions (Anprotec). HCMV-infected MRC-5 or HFF cells (MOI = 3, 72hpi) were incubated with titrated amounts of Cytotect at 37˚C for 1 hr in a 5% CO 2 atmosphere. After washing 2Â with medium, 5 Â 10 5 PBMCs were incubated on opsonized infected cells for 6 hr (100 ml per well in RPMI/10% FCS) in the presence of aCD107a-, aCD56-BV650, and Golgi-Plug/Golgi-Stop (according to supplier, BD). After incubation, PBMCs were harvested, washed in staining buffer (PBS/3% FCS), and incubated with aCD3-FITC 30 min at 4˚C. After two final washing steps in staining buffer, analysis was performed on a FACS Fortessa instrument (BD Bioscience).

Statistical analyses
Statistical analyses were performed using ANOVA or t-test (Prism5, Graphpad). Multiple comparison was corrected by Tukey test. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for all pertinent Figures (Figures 1D, 3D, 4C, 5D, 6B, 6C, 6D, Figure 4-figure supplement 1).