Fig 2: AKT1 interaction with UPF1 requires UPF3X. A proximity ligation assay was performed to assess the interactions between AKT1 and UPF1 or UPF3X after downregulation of UPF1 or UPF3X with siRNA. The white arrows indicate typical interaction points. The white squares correspond to a magnification of the background image. The bar plot at the bottom of the figure shows the average number of interaction points per cell as determined for >200 cells for each condition. Error bar = S.D., P-values were calculated with Student's t-test: *<0.05, **<0.01. All the results of this figure are representative of two experiments.

Fig 3: The capsid protein of ZIKV interacts with the NMD pathway. (a) Ugandan ZIKV capsid (Ug Cap) (MR 766) and French Polynesian ZIKV capsid (Fp Cap) (H/PF/2013) PPI maps that show significant enrichment for host NMD-associated factors (purple), as identified by AP-MS (SAINTq probability score of > 0.9 and false-discovery rate [FDR] of <0.05). Ten interactions between Fp Cap and host NMD factors (hypergeometrical test, P value = 7.16 × 10−10) and eight interactions between Ug Cap and host NMD factors (P value = 3.45 × 10−7) were identified. (b) Coimmunoprecipitation (co-IP) and Western blot analysis of HEK293T cells transfected with vector or Flag-tagged ZIKV capsid (H/PF/2013, Asian lineage) and harvested at 48 hpt to immunoprecipitate endogenous UPF3B. The upper band detected in the IP Capsid blot represents a nonspecific artifact. α-Flag, anti-Flag antibody. (c) Co-IP and Western blot analysis of HEK293T cells transfected with vector or Flag-tagged ZIKV capsid and harvested at 48 hpt to immunoprecipitate endogenous UPF1. (d) Myc tag co-IP and Western blot analysis of HEK293T cells transfected with Strep-tagged ZIKV capsid and Myc-UPF1 (wild type), Myc-UPF1-C126S (RNA-binding mutant) or Myc-UPF1-G495R/G497E (ATPase/helicase mutant) and harvested at 48 hpt to immunoprecipitate ZIKV capsid. (e) Western blot analysis of UPF1 levels in mock-infected and ZIKV-infected (PRVABC59, MOI of 1) Huh7 cells or mock-infected and ZIKV-infected (P6-740, MOI of 1) NPCs harvested at 48 hpi, with β-actin and ZIKV envelope (ZIKV E) or ZIKV capsid (ZIKV C) protein serving as loading and infection controls, respectively. Densitometric analyses were performed using ImageJ to quantify relative band intensities. Data are represented as means plus SEM. The P values were calculated by unpaired Student’s t test. **, P ≤ 0.01; ***, P ≤ 0.001. n = 3 independent experiments.

Fig 4: AKT1 interacts with NMD factors. (A) AKT1 immunoprecipitation analysis. AKT1 was immunoprecipitated from HEK293FT WT cells. The immunoprecipitations were performed in the presence of RNase A or BSA to assess the requirement for RNA in the protein interactions. The immunoprecipitate was then analyzed by western blotting for the presence of co-immunoprecipitated factors. MDM2 as a partner of AKT1 was used as positive control. (B) UPF1 immunoprecipitation analysis. UPF1 was immunoprecipitated from HEK293FT WT cells and the immunoprecipitate analyzed by western blotting for the presence of co-immunoprecipitated factors. (C) UPF1 immunoprecipitation analysis under NMD activation. UPF1 was immunoprecipitated from HEK293FT WT cells transfected with a construct expressing PTC-carrying globin mRNA in the presence of RNase A or BSA to assess the requirement for RNA in the protein interactions. The immunoprecipitate was then analyzed by western blotting for the presence of co-immunoprecipitated factors. The right panel is the result of an RT-PCR performed on RNA extracted from the immunoprecipitates to assess the efficiency of the RNase A treatment. (D) UPF3X immunoprecipitation analysis. UPF3X was immunoprecipitated from HEK293FT WT cells and the immunoprecipitate analyzed by western blotting for the presence of co-immunoprecipitated factors. GFP immunoprecipitation was performed as a negative control to assess the specificity of the immunoprecipitations. The immunoprecipitations were performed in the presence of RNase A or BSA to assess the requirement for RNA in the protein interactions. The immunoprecipitate was then analyzed by western blotting for the presence of co-immunoprecipitated factors. The right panel is the result of RT-PCR performed on RNA extracted from the immunoprecipitates to assess the efficiency of RNase A treatment. The three leftmost lanes in each panel of the figure correspond to serial dilutions of HEK293FT whole cell extract. The results presented in this figure are representative of two experiments.

Fig 5: AKT1 interacts with UPF1 and UPF3X. A proximity ligation assay was performed to assess interactions between AKT1 and UPF1, UPF2, UPF3X or MDM2 (positive control). As a negative control, interactions between UPF1 and eIF4E were assessed. The white arrows indicate typical interaction points. The white squares correspond to a magnification of the background image. The bar plot at the bottom of the figure shows for each condition the average number of interaction points per cell as determined on more than 200 cells for each condition. Error bar = S.D., P-values were calculated with Student's t-test: *<0.05, **<0.01. All the results of this figure are representative of two experiments.