Identification and Functional Characterization of a Novel Fc Gamma-Binding Glycoprotein in Rhesus Cytomegalovirus

Abstract

Receptors recognizing the Fc part of immunoglobulin G (FcγRs) are key determinants in antibody-mediated immune responses. Members of the Herpesviridae interfere with this immune regulatory network by expressing viral FcγRs (vFcγRs). Human cytomegalovirus (HCMV) encodes four distinct vFcγRs that differ with respect to their IgG subtype specificity and their impact on antibody-mediated immune function in vitro. The impact of vFcγRs on HCMV pathogenesis and immunomodulation in vivo is not known. The closest evolutionary animal model of HCMV is rhesus CMV (RhCMV) infection of rhesus macaques. To enable the characterization of vFcγR function in this model, we studied IgG binding by RhCMV. We show that lysates of RhCMV-infected cells contain an IgG-binding protein of 30 kDa encoded by the gene Rh05 that is a predicted type I glycoprotein belonging to the RL11 gene family. Upon deletion of Rh05, IgG-Fc binding by RhCMV strain 68-1 is lost, whereas ectopic expression of Rh05 results in IgG binding to transfected cells consistent with Rh05 being a vFcγR. Using a set of reporter cell lines stably expressing human and rhesus FcγRs, we further demonstrate that Rh05 antagonizes host FcγR activation. Compared to Rh05-intact RhCMV, RhCMVΔRh05 showed an increased activation of host FcγR upon exposure of infected cells to IgG from RhCMV-seropositive animals, suggesting that Rh05 protects infected cells from opsonization and IgG-dependent activation of host FcγRs. However, antagonizing host FcγR activation by Rh05 was not required for the establishment and maintenance of infection of RhCMV, even in a seropositive host, as shown by the induction of T cell responses to heterologous antigens expressed by RhCMV lacking the gene region encoding Rh05. In contrast to viral evasion of natural killer cells or T cell recognition, the evasion of antibody-mediated effects does not seem to be absolutely required for infection or reinfection. The identification of the first vFcγR that efficiently antagonizes host FcγR activation in the RhCMV genome will thus permit more detailed studies of this immunomodulatory mechanism in promoting viral dissemination in the presence of natural or vaccine-induced humoral immunity. IMPORTANCE Rhesus cytomegalovirus (RhCMV) offers a unique model for studying human cytomegalovirus (HCMV) pathogenesis and vaccine development. RhCMV infection of nonhuman primates greatly broadened the understanding of mechanisms by which CMVs evade or reprogram T cell and natural killer cell responses in vivo. However, the role of humoral immunity and viral modulation of anti-CMV antibodies has not been studied in this model. There is evidence from in vitro studies that HCMVs can evade humoral immunity. By gene mapping and with the help of a novel cell-based reporter assay system we characterized the first RhCMV encoded IgG-Fcγ binding glycoprotein as a potent antagonist of rhesus FcγR activation. We further demonstrate that, unlike evasion of T cell immunity, this viral Fcγ receptor is not required to overcome anti-CMV immunity to establish secondary infections. These findings enable more detailed studies of the in vivo consequences of CMV evasion from IgG responses in nonhuman primate models.

Human Cytomegalovirus antagonizes activation of Fcγ receptors II and III by distinct and synergizing modes of IgG manipulation

“…However, it is already known that HCMV vFcγRs also antagonize activation of FcγRIIA (CD32) and FcγRI (CD64) (Corrales-Aguilar, Trilling, et al, 2014) and we also consistently observed antagonization of the only inhibitory FcγR, FcγRIIB (unpublished observation). Similarly, we could recently show antagonization of all canonical Rhesus FcγRs I, IIA, IIB and III by the RhCMV encoded vFcγR 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 FcγRs IIA/B and III, we also speculate that more Fcγ binding host factors might be antagonized by similar mechanisms involving vFcγRs.…”

“…S4). Similar membrane-distal YXXΦ motifs also exist in the cytosolic domains of HCMV encoded gpRL13 and gB, making the di-leucine motif found in gp34 unique among other surface-resident glycoproteins expressed by HCMV and all other vFcγRs known in CMV family members including RhCMV and MCMV(Kolb et al, 2019; Thale, Lucin, Schneider, Eggers, & Koszinowski, 1994). It remains to be explored as to what the further consequences of this seemingly unique internalization route is.…”

“…Looking closely at the cytosolic domains of all identified cytomegalovirus vFcγRs reveals a unique [D/E]XXX[LL/LI] cytosolic motif present in gp34. Conversely, the HCMV FcγRs gp68, gp95 and gpRL13 all share only a cytosolic YXXΦ motif, as the recently identified RhCMV encoded vFcγR 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 YXXΦ motif of gp68 fulfills a more general purpose of limiting the overall surface exposure of concerned HCMV antigens (examples listed in Fig.…”

The Guinea pig cytomegalovirus GP119.1 gene encodes an IgG‐binding glycoprotein that is incorporated into the virion

“…Recently, it has also been demonstrated that rhesus CMV (RhCMV) Rh05 encodes a homolog of HCMV gp34 and mediates the IgG-dependent activation of rhesus FcγRs. 17 Our study has a limitation in the lack of demonstration of IgG-binding capacity of, or inhibition of, FcγR activation by GP119.1 in infected F I G U R E 5 Localization of GP119.1 expressed in the GPCMV-infected cells. At 90 h after infection of GPL cells with f119 or d119, the cells were stained with anti-DDDDK antibody against the FLAG tag, and fluorescent images were captured by a confocal microscope.…”

In vitroandin vivocharacterization of a recombinant rhesus cytomegalovirus containing a complete genome

“…Recombinant RhCMV clones were generated by en passant mutagenesis, as previously described for HCMV (65), and adapted by us for RhCMV (66). This technique allows the generation of fusion protein, have been described before (14,53).…”