Fig. 2. Rh173 binds IgG and antagonizes FcγR activation.

A 293 T cells transfected with pIRES-eGFP plasmids providing a Tapasin signal peptide and an N-terminal HA-tag encoding either an established vFcγR, an RhCMV RL11 family member or the human CD99 protein as a negative control were probed for binding of PE-conjugated rhesusized IgG1 with or without permeabilization via flow cytometry. The presented histograms show a full panel of vFcγRs and a CD99 control to illustrate IgG binding ranges. The bar graphs focus on RL11 family members without known IgG binding. Symbols show the mean fluorescence intensity (MFI) of independent experiments for IgG-binding (left panel) or MFI of protein expression using the N-terminal HA-tag (right panel), both normalized to UL119/118 signals. Bars show the mean of independent experiments. Empty gray bars represent RL11 family members that lack a predicted signal peptide and/or transmembrane domain in their original sequence. B HeLa cells were transfected with plasmid pIRES-eGFP expressing a T2A-linked fusion protein of the RhCD4 target antigen, and the indicated Rh173 sequence variants from published RhCMV sequences. The transfectants were incubated with graded amounts of RhCD4-specific rhesusized IgG1 and tested for human CD16 activation using a cell-based FcγR activation reporter assay. Equal transfection efficiency was monitored via polycistronic GFP expression. A non-Fcγ binding glycoprotein control (CD99) served as control. Symbols show mean area under curve (AUC) values of independent experiments performed in technical replicates. Bars show the mean of independent experiments. Source data are provided as a Source Data file.