The GP1-binding site on human TfR1 (hTfR1) has been pinpointed to a prominent loop within the apical domain (residues 201-212) [22] . Conversely, until recently the TfR1-binding site on arenavirus GP1 has been poorly characterized. One study showed that the 20 N-terminal amino acids of MACV GP1 are dispensable for TfR1-binding as a MACV GP1 variant lacking these residues (MACV GP1D, residues 79-258) binds hTfR1 even more efficiently than wild-type GP1 [17] . A second study demonstrated that the central region of GTOV GP1 (residues 85-221) interacts with hTfR1 and that residues 159-221 are essential for this interaction [23] . A recently published crystal structure of a truncated MACV GP1 (region 87-239) [24] , as well as a co-crystal structure of the MACV GP1:TfR1 complex [25] provided structural insight of GP1 residues that contact TfR1. The latter study identified five interaction motifs in the interface between MACV GP1 and the apical domain of TfR1 involving some relatively conserved GP1 residues, such as D114 (motif 2), D123 (motif 4), and F226 (motif 3) (colored red, Fig. 1 ), some less conserved residues, such as Y122 (motif 4) and K169 (motif 4) (colored blue, Fig. 1 ), as well as some nonconserved residues, such as R111 (motif 1),and S116 (motif 2) (colored green, Fig. 1 ) [25] . To evaluate experimentally the functional significance of these residues and other conserved solvent-accessible MACV GP1 residues in TfR1-mediated entry, we created a panel of MACV GP1 variants replacing the mentioned amino acids with alanine, and tested the variants' ability to bind hTfR1 and mediate MACV entry in simian and human cells experimental systems.