Fig. 4: RhCMV vFcγRs mirror HCMV vFcγR function and efficiently antagonize host FcγR activation in vitro.

A Graphical overview of HCMV vFcγRs and their RhCMV orthologs. Created in BioRender. Kolb, P. (2024) https://BioRender.com/f19l834. B Transfected HeLa cells expressing both the rhesus CD4 target antigen and the indicated vFcγRs as T2A-linked fusion protein from a pIRES-e-GFP plasmid were incubated with graded amounts of a rhesus CD4-specific rhesusized IgG1 antibody and tested for human CD16 activation using a cell-based reporter assay. Equal transfection was monitored via polycistronic GFP expression. Empty vector (e.v.) transfection and expression of a non-Fcγ binding glycoprotein (CD99) served as controls. Symbols show mean area under curve (AUC) values of independent experiments. Bars show the mean of independent experiments. C Overview of the RhCMV 68-1 based recombinants. To create RhCMV 68-1 R, we repaired a premature termination codon in Rh152/151. Single and triple deletion mutants of the vFcγRs were based on the repaired 68-1 strain. All deletions are indicated by highlighting the corresponding ORFs in red. D Telomerized rhesus fibroblasts (tRF) were infected with RhCMV 68-1 or 68-1 R or 68-1 R derived single vFcγR deletion mutants at an MOI of 5 for 72 h and then labeled with [³⁵S]-Met/Cys for 2 h. Whole-cell lysates were prepared, and precipitations of IgG binding molecules were performed using ProteinG bound rhesus-CD4 specific rhesusized IgG1. Lysates from each condition were split and either left untreated or digested with either EndoH or PNGaseF. All samples were analyzed via gradient SDS-PAGE and subsequent autoradiography. One of two independent experiments is presented here. E RFs were infected with RhCMV 68-1, 68-1 R, or single vFcγR deletion mutants at an MOI of 2 for 48 h and then incubated with a 1:10 dilution of a pooled sera from eight CMV seropositive RM. The samples were assessed for human or rhesus FcγR activation using a cell-based reporter assay. The resulting mIL-2 ELISA OD values were normalized to the mean activation on the vFcγR deleted virus for each subgroup. vFcγRs expressed by viruses used for infection are indicated below. FL-RhCMV infection is indicated in pink, and complete vFcγR deletion is indicated in green. * = mock-infected cells. Symbols show independent experiments performed in technical replicates. Bars show the mean of independent experiments. Source data are provided as a Source Data file.