Fig. 3: AMFR mediates flavivirus-NS2A ubiquitination which is required for NS2A-drived FAM134B degradation.

a, b, Western blotting analysis of HEK293T cells transfected for 30 h with the indicated plasmids and then treated with DMSO, MG132 (10 μM), and CQ (50 μM) for 8 h (a); Quantification of relative FAM134B level (b). c–f, h, j, HEK293T cells were transfected with the indicated plasmids. Cells were harvested for coIP using anti-Flag beads and analyzed by western blotting with a K48-linkage-specific polyubiquitin antibody to detect the ubiquitination of NS2A. g In vitro ubiquitination assays were measured by incubating purified Flag-NS2A with purified HA-AMFR_WT or HA-AMFR_C356G/H361A analyzed by western blotting with a K48-linkage specific polyubiquitin antibody to detect the ubiquitination of NS2A. i Huh7.0 cells were either mock-infected or infected with ZIKV-HA-NS2A at an MOI of 1 for 72 h and then cells were harvested for coIP using anti-HA beads and analyzed by western blotting. k A549 and Vero cells were mock-infected or infected with ZIKV-HA-NS2A_WT (MOI = 0.1) or ZIKV-HA-NS2A_K56R (MOI = 1) for 72 h and then harvested for coIP using anti-HA beads and analyzed by western blotting with a K48-linkage specific polyubiquitin antibody to detect the ubiquitination of NS2A and the input E was normalized for immunoprecipitation. l, m Western blotting analysis of HEK293T cells or HEK293T-AMFR knockdown cells transfected for 48 h with the indicated plasmids (l); Quantification of relative FAM134B level (m). n Huh7.0-GFP-FAM134B_R142A cells were infected with ZIKV-NS2A_WT (MOI = 1), ZIKV-NS2A_K56R (MOI = 10), or mock-infected for 72 h, determined by flow cytometry. Error bars of (b, m, n) present as the mean ± SEM (n = 3 independent experiments); Statistical analysis of (b) was performed using two-way ANOVA with Sidak’s multiple comparisons test and (m, n) was performed using one-way ANOVA with Tukey’s multiple comparisons test; Source data are provided as a Source Data file.