Fig 2: Plxdc family receptors are novel interaction partners for RRV gH/gL.A) Immunoprecipitation of recombinant soluble RRV 26–95 gH-FcStrep in the presence or absence of 293T lysate. Precipitates were analyzed by PAGE, silver stained and bands at the indicated molecular weight (arrows, regions a-e) were excised and analyzed by mass spectrometry. Numbers in brackets indicate the number of Plxdc2 peptides identified by LC-MS/MS in each region. B) V5-tagged RRV 26–95 gH, RRV 17577 gH or KSHV gH alone or co-expressed with the respective Flag-tagged gL construct were immunoprecipitated in the presence of full-length Plxdc1 or Plxdc2 of human (h) or Macaca mulatta (mm) origin using monoclonal antibody to the V5-tag. Precipitates were analyzed by Western blot with the indicated antibodies. C-D) Binding of Plxdc1 ectodomain C) or Plxdc2 ectodomain D) at various concentrations to immobilized RRV 26–95 gH-FcStrep/gL was measured by enzyme-linked immunosorbent assay. Curve Fitting and determination of half-maximal binding concentration was performed based on the One site specific binding with Hill Slope equation in Prism6. E) Co-immunoprecipitation of soluble human Plxdc1-FcStrep or human Plxdc2-FcStrep with RRV 26–95 gH-V5/gL-Flag or RRV 17577 gH-V5/gL-Flag using StrepTactin sepharose in the presence or absence of human full-length myc-tagged EphB3. Abbreviations: IP: immunoprecipitation, IB: immunoblotting, h: human, mm: Macaca mulatta (rhesus macaque).

Fig 3: Plxdc1/2 function as entry receptors for RRV.A) List of BAC-derived recombinant viruses and introduced mutations used in this figure. B) Dose-dependent inhibition of RRV 26–95 infection by soluble human Plxdc2-FcStrep on HaCaT cells. RRV-YFP wt and RRV-YFP gH-AELAAN were pre-incubated with hPlxdc2-FcStrep for 30min at room temperature. FcStrep alone was used as control. YFP expression as indicator of infection was measured by flow cytometry. Infection in the presence of 0.4nM FcStrep was set to 100% (MOI ~0.2, triplicates, error bars represent SD). C) Inhibition of RRV 26–95 infection by soluble Plxdc2-FcStrep and EphB3-Fc on HaCaT cells. RRV-YFP wt and RRV-YFP gH-AELAAN were pre-incubated with 100nM hPlxdc2-FcStrep, 10nM EphB3-Fc or a combination of 100nM hPlxdc2-FcStrep and 10nM EphB3-Fc for 30min at room temperature. FcStrep alone was used as control. YFP expression as indicator of infection was measured by flow cytometry. Infection with FcStrep was set to 100% (MOI ~0.2, triplicates, error bars represent SD). D) Inhibition of RRV 26–95 infection by soluble human Plxdc2-FcStrep on SLK cells and rhesus monkey fibroblasts (RF). RRV-YFP wt and RRV-YFP gH-AELAAN were pre-incubated with 250nM hPlxdc2-FcStrep for 30min at room temperature. FcStrep alone was used as control. YFP expression as indicator of infection was measured by flow cytometry. Infection with FcStrep was set to 100% (MOI ~0.05–0.1, triplicates, error bars represent SD). E-F) Raji cells were transduced with TwinStrep-tagged human Plxdc1, Plxdc2 or EphA7 (hPlxdc1-Strep/ hPlxdc2-Strep/ hEphA7-Strep) expression constructs or an empty vector control, briefly selected and infected with RRV-YFP 26–95 wt and RRV-YFP 26–95 gH-AELAAN (E) or RRV-YFP 17577 (F) normalized to genome copies as determined by qPCR. Micrographs show representative infection of the indicated cell pools. G) Lysates of transduced Raji cell pools were analyzed for EphA7-Strep and Plxdc1/2-Strep expression by Western blot. H) Quantification of (E) by flow cytometric analyses of YFP reporter gene expression as indicator of infection (triplicates, error bars represent SD). I) Quantification of (F) by flow cytometric analyses of YFP reporter gene expression as indicator of infection (triplicates, error bars represent SD).