Four mutants, D114A, D140A, K167A, and K169A, precipitated hTfR1 as efficiently as wild-type (wt) MACV GP1D or at slightly lower levels. Reduction in GP1 interaction with hTfR1 was observed with mutants S116A, K211A, and D114A/S116A (motif 2 in [1] ), whereas mutants R111A, Y122A, D123A, W147A, D155A, D159A, P160A, N178A, F226A, and the triple mutant Y122A/D123A/K169A effectively inhibited MACV receptor interactions (Fig. 2B) . We performed cell-binding assays as described [17, 26] to determine the ability of these mutants to bind to the surface of MACV-permissive cells. Briefly, Fc constructs were added to 5610 5 Vero cells (ATCC) to a final concentration of 200 nM. Cells with bound proteins were incubated with a 1:40 dilution of FITC-conjugated anti-human Fc antibody (Sigma-Aldrich). Cell-surface binding of constructs was detected by flow cytometry. Baseline fluorescence was determined by measuring cells treated only with the secondary antibody, which was then subtracted from binding values of the tested constructs and control proteins. Overall, cell-binding of mutants supported the co-IP results as mutants that precipitated hTfR1 exhibited high cell-surface binding and vice versa (Fig. 2C) . Exceptions were Y122, S116A and D114A/S116A (motif 2), which bound the cell-surface of Vero cells as or almost as efficiently as wt MACV GP1D, yet exhibited lower hTfR1 levels in the co-IP assays. The K167A mutant exhibited an opposite trend, as it bound less efficiently to the cell-surface than wt MACV GP1D whereas it immunoprecipitated hTfR1 as effectively.