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. Author manuscript; available in PMC: 2021 May 18.
Published in final edited form as: Nature. 2020 Nov 18;588(7837):308–314. doi: 10.1038/s41586-020-2915-3

Figure 1. Ldlrad3 is required for efficient VEEV infection in cells.

Figure 1.

a. Enriched genes based on top p-values (top panel) or robust rank aggregation scores (bottom panel) in the SINV-VEEV selected population. b. ΔB4galt7 (control, black), ΔB4galt7 ΔLdlrad3 (ΔLdlrad3, red), and Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 (ΔLdlrad3 + Ldlrad3, blue) N2a cells were inoculated with SINV-VEEV-GFP (IAB strain TrD, IC strain INH9813, and ID strain ZPC738) or VEEV TC-83, and infection levels were assessed through GFP expression or E2 antigen staining. SINV-VEEV TrD (IAB), VEEV TC83, and SINV-VEEV (IC): n = 9; SINV-VEEV (ID): n = 8. Mean ± standard deviation (SD) of three experiments. c. Control (gray), ΔB4galt7 (black), ΔLdlrad3 (dark red), ΔB4galt7 ΔLdlrad3 (red), and Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 (blue) BV2 cells were inoculated with SINV-VEEV-GFP (strain TrD), and infection levels were assessed by flow cytometry. WT and ΔLdlrad3, n = 18; ΔB4galt7 and ΔB4galt7 ΔLdlrad3, n = 12; ΔB4galt7 ΔLdlrad3 + Ldlrad3, n = 6. Mean ± SD of three to six experiments. d. Multi-step growth curves of ΔB4galt7 (control, black), ΔB4galt7 ΔLdlrad3 (ΔLdlrad3, red), and Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 (ΔLdlrad3 + Ldlrad3, blue) N2a cells with SINV-VEEV viruses (IAB strain TrD, IC strain INH9813, and ID strain ZPC738). Mean ± SD of three experiments (n = 9). e. Control (black), ΔLdlrad3 (red), and Ldlrad3-complemented ΔLdlrad3 (blue) N2a cells retaining GAG biosynthesis (B4galt7+/+) were inoculated with SINV-VEEV-GFP (strain TrD), and infection was assessed by flow cytometry. Mean ± SD of three experiments (n = 9). f. ΔB4galt7 (control, black), ΔB4galt7 ΔLdlrad3 (red), and Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 (blue) N2a cells were inoculated with VEEV IAB TrD-GFP (left panel) or EEEV FL93-939-GFP (right panel) and assessed for infection by flow cytometry. Mean ± SD of two (EEEV) or three (VEEV) experiments (EEEV: n = 6; VEEV: n = 9). g-j. ΔB4galt7 (control, black), ΔB4galt7 ΔLdlrad3 (red), and Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 (blue) N2a cells were inoculated with SINV-EEEV or SINV-WEEV (g), SINV (strains AR86, TR339, Toto1101, or Girdwood) (h), MAYV (i), or VSV (j) and infection was assessed via GFP expression or viral antigen staining. (g) SINV-EEEV and SINV-WEEV, n = 9. (h) AR86 and Toto1101, n = 9; TR339 and Girdwood, n = 8. (i) MAYV, n = 9. (j) VSV, n = 9. Mean ± SD of three experiments. k. Multi-step growth curves of ΔB4galt7 (control, black), ΔB4galt7 ΔLdlrad3 (red), and Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 (blue) N2a cells with WNV-Kunjin. Mean of two experiments performed in duplicate. l. ΔB4galt7 (control, black), ΔB4galt7 ΔLdlrad3 (red), and LDLRAD3-complemented ΔB4galt7 ΔLdlrad3 (blue) N2a cells were inoculated with SINV-VEEV-GFP (strain TrD) and infection was assessed by flow cytometry. Mean ± SD of three experiments (n = 9). m. Control (black), ΔLDLRAD3 (red), full-length Ldlrad3-complemented (blue), and truncated Ldlrad3 isoform-complemented ΔLDLRAD3 (orange) B4GALT7+/+ human SH-SY5Y cells were inoculated with SINV-VEEV-GFP (strain TrD), and infection was assessed by flow cytometry. Mean ± SD of three experiments (n = 9). n. Multi-step growth curves of control (black) and ΔLDLRAD3 (red) B4GALT7+/+ human SH-SY5Y cells. Mean ± SD of three experiments (n = 9: two-way ANOVA with Dunnett’s post-test: **** P < 0.0001). One-way (e, f, l, m) or two-way (b, c, n) ANOVA with Dunnett’s post-test: **** P < 0.0001; ns, not significant. d, two-way ANOVA with Dunnett’s post-test: +++ P < 0.001; **** or ++++ P < 0.0001; ns, not significant. “*” [black] indicates a comparison between ΔB4galt7 [control] and ΔB4galt7 ΔLdlrad3 N2a cells (SINV-VEEV (IC): 12 h, P = 0.8935; SINV-VEEV (ID): 12 h, P = 0.0553). “+” [blue] indicates a comparison between Ldlrad3-complemented ΔB4galt7 ΔLdlrad3 and ΔB4galt7 ΔLdlrad3 N2a cells (SINV-VEEV (IC): 12 h, P = 0.8541; 24 h, P = 0.0003; SINV-VEEV (ID): 12 h, P = 0.6690).