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. Author manuscript; available in PMC: 2024 Oct 26.
Published in final edited form as: Cell. 2023 Oct 6;186(22):4818–4833.e25. doi: 10.1016/j.cell.2023.09.007

Figure 6. Chimeric avian-mammalian MXRA8 interacts with both WEE and SF complex alphaviruses.

Figure 6.

A. Schematic of chimeric MXRA8 proteins used to assess binding modes. In addition to mouse or duck MXRA8, chimeras shown are Du-D1-Mo-D2 and Mo-D1-Du-D2. B-C. SINV-GFP (B) and SINV-WEEV-GFP (C) infection in ΔMxra8 3T3 cells complemented with wild-type duck (blue) and chimeric (blue/pink) duck-mouse MXRA8 (3 experiments in triplicate; normalized to wild-type duck MXRA8 infection. Infection and GFP fluorescence were analyzed by flow cytometry. D-E. SINV-CHIKV (LR 2006 strain) (D) and MAYV (E) infection of ΔMxra8 3T3 cells complemented with an empty vector, or wild-type mouse and chimeric duck-mouse MXRA8 (3 experiments in triplicate; normalized to wild-type mouse MXRA8 infection). Infection was analyzed by flow cytometry by evaluating viral antigen staining with mAbs (CHIKV or MAYV). F-I. Staining of the surface of Vero cells infected with SINV-TR339 (F), SINV-CHIKV (LR 2006 strain), (G) MAYV (H), or SINV-VEEV (I) after incubation with serially diluted mouse MXRA8-Fc, duck MXRA8-Fc, Du-D1-Mo-D2 MXRA8-Fc, LDLRAD3-D1-Fc proteins or cross-reactive anti-E1 mAb (DC2.112, positive control)37) control. Data are expressed as the percentage of infected cells that bound positively to the indicated proteins by flow cytometry (representative of 2 experiments). J-L. Neutralization of SINV TRR399 (J) SINV-WEEV (K) or SINV-CHIKV (L) (all three viruses expressing eGFP) infection by duck MXRA8-Fc, mouse MXRA8-Fc, Du-D1-Mo D2-MXRA8-Fc, Mo-D1-Du-D2-MXRA8-Fc, or LDLRAD3-D1-Fc control (3 experiments, duplicates). Infection was analyzed by flow cytometry (GFP expression) and normalized to levels after incubation with LDLRAD3-D1-Fc protein. M-N. Clinical disease (M) and survival (N) of 4-week-old female CD-1 mice inoculated with 103 PFU of WEEV (McMillan strain) mixed with Du-D1-Mo D2 N66R-MXRA8-Fc or LDLRAD3-D1-Fc. O-S. Foot swelling (O) and viral RNA levels in indicated tissues (P-S) of 4-week-old male C57BL/6J mice at 72 h after inoculation with 103 FFU of CHIKV (La Reunion 2006) mixed with 50 μg of mouse Mxra8-Fc, Du-D1-Mo-D2-N66R-MXRA8-Fc, or LDLRAD3-D1-Fc (O: 2 experiments n = 6; P-S: 2 experiments n = 10). B-E and O: one-way ANOVA with Dunnett’s post-test; mean ± standard deviation (SD). N: Log rank test. P-S: Kruskal-Wallis ANOVA with Dunn’s post-test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. See also Fig S11, S12, and Table S5.