We followed a strategy previously employed for virus receptor identification to evaluate the importance for TfR1 binding of these conserved residues, as well as GP1 residues recently indicated in the published MACV GP1:TfR1 crystal structure [17, 26] . Briefly, we designed codon-optimized open reading frames (ORFs) of MACV GP1D in which one or more of the chosen residues were mutated to alanine, and generated them by gene synthesis (DNA2.0). These ORFs were cloned into a pCDM8-like expression vector [17, 26] with the signal sequence of human CD5 upstream and the Fc region of human immunoglobulin G1 downstream of the ORF. Fc alone and wild-type MACV GP1D-Fc, which was described previously [17] , served as controls. Proteins were purified as described [17, 26] : the individual plasmids were transfected into HEK 293T/17 cells (ATCC) using the calcium-phosphate method and grown in 293 SFM II medium (GIBCO-Invitrogen). Media were harvested and clarified, and Fc fusion proteins precipitated with protein A-sepharose Fast Flow beads (GE Healthcare). Fc fusion proteins were eluted with 3 M MgCl 2 , dialyzed in PBS, and concentrated. Purified proteins were assayed by SDS-PAGE followed by Bio-Safe Coomassie staining (BIO-RAD), and measured using the Quant-iT Protein Assay kit (Invitrogen). All mutant plasmids expressed proteins ( Fig. 2A) . We evaluated the ability of the individual mutants to bind to hTfR1 using co-immunoprecipitation (co-IP) assays as described [17, 22, 26] . Briefly, HEK 293T/17 cells were transfected with plasmid encoding hTfR1 [17, 22] and lysed 48 h post transfection with RIPA Lysis and Extraction buffer (Thermo Scientific Pierce). Cleared lysates were added to equimolar amounts (200 nM) of Fc, MACV GP1D, or variants thereof, and bound proteins were immunoprecipitated with protein A-sepharose Fast Flow beads. hTfR1 in the cell lysate immunoprecipitates was analyzed by SDS-PAGE and western blot using the WesternBreeze Chromogenic kit (Invitrogen) and a murine monoclonal anti-TfR1 antibody (Invitrogen).