Fig. 4: FrMLV-specific recognition of mCAT1.
From: Structural insights into cationic amino acid transport and viral receptor engagement by CAT1
a Multiple-sequence alignment of mCAT1 from various species (Mus musculus mCAT1 (Q09143), Rattus norvegicus rCAT1 (P30823), Homo sapiens hCAT1 (P30825), Pan troglodytes pCAT1 (H2Q7D2), Equus caballus eCAT1 (F6TT99), Ailuropoda melanoleuca aCAT1 (D2H021)) showing conserved residues and cysteines (C226 and C309) that form the ECL3–ECL4 disulfide. b Mapping of ECL3 (blue) and ECL4 (magenta) on the mCAT1/frRBD structure, highlighting key residues involved in receptor binding (F224, F242–F244, Y235, C226, C309). c Pseudovirus entry assays in Lenti-X 293 T cells stably expressing the indicated receptors. Swapping ECL3 between mouse and human reciprocally modulates infection: mCAT1 supports high entry, hCAT1 does not, mCAT1–hloop (mouse backbone bearing human ECL3) shows markedly reduced entry, whereas hCAT1–mloop becomes permissive. Bars show mean relative luminescence units (RLU, log scale) ± s.e.m.; individual biological replicates are shown as dots; n is indicated above bars. Two-sided Student’s t-test versus the relevant control: ****P < 0.0001. d Effect of disrupting the ECL3–ECL4 disulfide on infection. Pseudovirus entry mediated by C309S is moderately reduced relative to WT mCAT1; NC, negative control. Bars, mean RLU (log) ± s.e.m.; n as indicated; **P < 0.01 (two-sided t-test). e Biolayer interferometry (BLI) sensorgrams for frRBD binding to the mCAT1 (C309S). frRBD concentrations (40–1,280 nM) are color-coded; y-axis, response (nm); x-axis, time (s). Global fitting yielded KD = 294 ± 4.4 nM. f Entry mediated by FrMLV variants. Pseudoviruses bearing FrMLV (isolate 57) and Moloney murine leukemia virus (MoMLV) show comparable infection in mCAT1-expressing cells (mean RLU (log) ± s.e.m.; n as indicated). (c, d, f) Data represent n = 12 biological samples derived from three independent experiments (4 independent samples per experiment). c, d Exact P values: mCAT1 vs hCAT1: P < 0.0001; mCAT1-hLoop vs hCAT1-mLoop: P < 0.0001; WT vs C309S: P = 0.0476. g Sequence-logo representation of Env residues at the receptor-binding interface compiled from FrMLV subtypes and additional MLV lineages. High conservation is observed across interface positions; red arrowheads mark residues implicated in receptor contacts in this study.
