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. 2024 Nov 20;13:RP94347. doi: 10.7554/eLife.94347

Figure 4. IGF2BP2 associates with NS5 and accumulates into Zika virus (ZIKV) replication compartment.

(A) Huh 7.5 cells were infected with ZIKV H/PF/2013 at an MOI of 5. Cells were collected at 48 and 72 hr post-infection (hpi). Cell extracts were prepared and analyzed by western blotting using the indicated antibodies. Actin-normalized protein signals are shown. (B) Huh7.5 cells were infected with ZIKV H/PF/2013 with an MOI of 10 or left uninfected. Two days post-infection, cells were fixed, immunolabeled for the indicated factors, and imaged by confocal microscopy. Scale bar = 10 µm. The Manders’ coefficient (mean ± SEM) representing the fraction of dsRNA (cyan) and NS3 (red) signals overlapping with IGF2BP2 signal is shown (n=number of cells). (C) Co-immunoprecipitation assays using HA antibodies were performed with extracts from Huh7.5 cells stably expressing IGF2BP2-HA (+) or control-transduced cells (-) which were infected with ZIKV at an MOI of 10 for 2 days. Purified complexes were analyzed for their protein content by western blotting. (D) Means of quantified NS5 signals from (C) (normalized to actin [extracts] or IGF2BP2 [IP]) ± SEM are shown based on nine independent experiments. ****: p<0.0001; ns: not significant (unpaired t-test).

Figure 4—source data 1. Data points to generate the bar graphs of Figure 4D and quantify mean Manders’ coefficients of Figure 4B.
Figure 4—source data 2. PDF file containing original western blots for Figure 2A and C, indicating the relevant bands and conditions.
Figure 4—source data 3. Original files for western blot analysis displayed in Figure 2A and C.

Figure 4.

Figure 4—figure supplement 1. IGF2BP2 relocalizes to the viral replication compartment in Zika virus (ZIKV)-infected cells at 1 day post-infection.

Figure 4—figure supplement 1.

Huh7.5 cells were infected with ZIKV H/PF/2013 with an MOI of 10 or left uninfected. At 1 day post-infection, cells were fixed, immunolabeled for the indicated factors, and imaged by confocal microscopy. Scale bar = 10 µm. The white squares indicate the magnified areas in the insets.
Figure 4—figure supplement 2. HA-tagged IGF2BP2 relocalizes to the viral replication compartment in Zika virus (ZIKV)-infected cells, as endogenous IGF2BP2.

Figure 4—figure supplement 2.

Huh7.5 cells stably overexpressing IGF2BP2-HA or control-transduced cells (pWPI) were infected with ZIKV H/PF/2013 with an MOI of 10 or left uninfected. Two days post-infection, cells were fixed, immunolabeled for the indicated factors, and imaged by confocal microscopy. Scale bar = 10 µm. The white squares indicate the magnified areas in the insets.