A RNAi mini-screening of RNA-binding proteins to identify host factors involved in DENV and ZIKV replications.

Huh7.5 were transduced with shRNA-expressing lentiviruses at a MOI of 5-10. (A) Four days posttransduction, MTT assays were performed to evaluate cytotoxicity effect of the KD. Two days posttransduction cells were infected with either (B) ZIKV H/PF/2013, (C) ZIKV MR766, or (D) DENV2 16681s at an MOI of 0.01. 48h post infection, the production of infectious viral particles was evaluated by plaque assays. (E) Schematic of the Rluc-expressing ZIKV reporter virus (ZIKV-R2A) based on the FSS13025 isolate (Asian lineage). (F) Cells were prepared, exactly as in B-D but infected with ZIKV-R2A at a MOI of 0.001. 48h post-infection, cells were lysed and bioluminescence was measured and normalized to the control cells expressing a non-target shRNA (shNT). Means ± SEM are shown based on three to five independent experiments for each shRNA. p<0.0001; ***: p< 0.001; **: p < 0.01; * p< 0.05 (one-way ANOVA test).

IGF2BP2 positively regulates ZIKV replication in multiple cell lines.

Liver Huh7.5 (A-C), astrocytic NHA-hTERT (D) and placental JEG-3 (E) cells were transduced with shNT or shIGF2BP2 lentiviruses at a MOI of 10. Two days post transduction, cells were infected with ZIKV H/PF/2013 at a MOI between 0.01 and 1 depending on the cell line. Two days post-infection, supernatant and cells were collected. IGF2BP2 expression at the protein level (A, D, E; all cell lines) and mRNA level (B; Huh7.5 cells) were evaluated by western blotting and RT-qPCR, respectively. Cell supernatants were used for plaque assays (C-E). for NHA-hTERT and JEG-3, the supernatant and cells are collected for titration and WB respectively (E-F). MTT assays were performed to assess the cell viability in transduced NHA-hTERT and JEG-3 cells (D-E). Means ± SEM are shown based on five (D), three (C) and four (D-E) independent experiments. ****: p<0.0001; ***: p< 0.001; **: p < 0.01 (unpaired t-test).

IGF2BP2 dependency is ZIKV-specific.

Huh7.5 cells were transduced with shNT or shIGF2BP2 lentiviruses at a MOI of 10. Two days post-transduction cells were infected with (A) ZIKV FSS13025, (B) DENV1 HAWAII, (C) DENV2 NGC, (D) DENV3 H84, (E) DENV4 H241, (F) WNV NY99, (G) SARS-CoV-2 at a MOI of 0.1. Virus-containing cell supernatants were collected and titrated two-days post-infection by plaque assays. Treatment with RdRp inhibitors NITD008 and Remdesivir were used as positive controls of replication inhibition of orthoflaviviruses and SARS-CoV-2, respectively. Means ± SEM are shown based on three independent experiments. ****: p<0.0001; *: p < 0.05; NS: not significant (unpaired t-test).

IGF2BP2 associates with NS5 and accumulates into ZIKV replication compartment.

(A) Huh 7.5 cells were infected with ZIKV H/PF/2013 at a MOI of 5. Cells were collected at 48-and 72-hours postinfection (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 a MOI of 10 or left uninfected. Two days post-infection, cells were fixed, immunolabelled for the indicated factors, and imaged by confocal microscopy. Scale bar=10 m). 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 a MOI of 10 for 2 days. Purified complexed 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).

IGF2BP2 interacts with ZIKV vRNA.

(A) FISH and IGF2BP2 immunostaining were performed using Huh 7.5 cells which were infected for 2 days with ZIKV (MOI = 10) or left uninfected, The Manders’ coefficient (mean ± SEM) representing the fraction of vRNA signal overlapping with IGF2BP2 signal is shown (n=number of cells). (B) Huh7.5 cells expressing IGF2BP2-HA and control cells were infected with ZIKV H/PF/2013 at a MOI of 10, or left uninfected. Two days later, cell extracts were prepared and subjected to anti-HA immunoprecipitations. Extracted vRNA levels were measured by RT-qPCR. Means ± SEM are shown based on three independent experiments. ****: p<0.0001; NS: not significant (unpaired t-test). (C) IGF2BP2 recombinant proteins containing either the two RRM or KH3 and KH4 domains were produced in bacteria and purified. In parallel ZIKV 5’ NTR and 3’ NTR were synthesized by in vitro transcription. (D) Combination of truncated IGF2BP2 proteins and either ZIKV 5’ NTR (blue squares) or ZIKV 3’ NTR (green circles) were used for in vitro binding assays using microscale thermophoresis.

IGF2BP2 regulates the replication of ZIKV vRNA.

(A) Schematic representation of reporter ZIKV H/PF/2013 sub-genomic replicons (sgR2A) and replication-deficient genomes because of mutations in NS5 RdRp sequence (sgR2A GAA). (B-C) Huh7.5 were transduced with shRNA-expressing lentiviruses and subjected to electroporation with in vitro transcribed sgR2A or sgR2A GAA RNAs two days later. In-cell bioluminescence was measured (B) 48 or (C) 4 hours post-electroporation and normalized to the shNT control condition. In C, the luciferase activity was normalized to the transfection efficiency i.e., the Rluc activity at 4h post-electroporation. Means ± SEM are shown based on four independent experiments. ***: p<0.001; NS: not significant (unpaired t-test).

ZIKV infection decreases the interaction between IGF2BP2 and several of its mRNA endogenous ligands.

Huh7.5 cells stably expressing IGF2BP2-HA (+) and control cells (-) were infected with ZIKV H/PF/2013 at a MOI of 10, or left uninfected. Two days later, cell extracts were prepared and subjected to anti-HA immunoprecipitations. Extracted (A) TNRC6A, (B) PUM2, and (C) CIRBP mRNA levels were measured by RT-qPCR. Means ± SEM are shown based on three independent experiments. ****: p<0.0001; ***: p<0.001; NS: not significant (unpaired t-test).

ZIKV infection does not significantly impact the association of IGF2BP2 with IGF2BP1, IGF2BP3 and YBX1.

Huh7.5 cells stably expressing IGF2BP2-HA (+) and control cells (-) were infected with ZIKV H/PF/2013 at a MOI of 10, or left uninfected. Two days later, cell extracts were prepared and subjected to anti-HA immunoprecipitations. (A) Purified complexes were analyzed by western blotting for their content in the indicated proteins. IGF2BP1 (B), IGF2BP3 (C) and YBX1 (D) levels were quantified and means of protein signals (normalized to actin (extracts) and IGF2BP2 (IP)) ± SEM are shown based on six to eight independent experiments. ns: not significant (unpaired t-test).

ZIKV infection alters the IGF2BP2 proteo-interactome.

Huh7.5 cells expressing IGF2BP2-HA and control cells were infected with ZIKV H/PF/2013, DENV2 16681s, or left uninfected. Two days later, cell extracts were prepared and subjected to anti-HA immunoprecipitations. Resulting complexes were analyzed by quantitative mass spectrometry. (A) Venn diagram depicting the overlap between IGF2BP2 partners modulated by ZIKV and/or DENV infections. (B) Gene Ontology (GO) biological process analyses of the IGF2BP2 interactions which were impacted upon ZIKV infection. (C) Interaction tree of the 62 IGF2BP2 interactions modulated by ZIKV infection (generated with STRING online resource). The red and green circles identify the partners of the STRING network whose association with IGF2BP2 is decreased and increased during infection, respectively. The biological process analysis generated by STRING is also shown.

IGF2BP2 regulates the biogenesis of ZIKV replication organelles.

(A) Schematic representation of the pIRO system. Upon transfection in cells expressing the T7 RNA polymerase, this plasmid allows the cytoplasmic transcription of NS1-NS5 polyprotein under the control of T7 promoter, in a ZIKV replication-independent manner. NS1-5 polyprotein synthesis is under the control of ECMV IRES. The presence of both ZIKV 3’ NTR and 5’ cyclization sequence (5’ CS) is required for efficient VP induction. Finally, the activity of HDV ribozyme ensures that the 3’ terminus of the RNA is similar to that of vRNA genome. Huh7-Lunet-T7 were transduced with shRNA-expressing lentiviruses at a MOI of 5-10. Two days later, transduced cells were transfected with pIRO-Z plasmid. Sixteen hours later, cells were analyzed for (B) IGF2BP2 mRNA levels by RT-qPCR to measure knockdown efficiency, (C-D) transfection efficiency by confocal imaging of NS3-labeled cells, and (E) for VP content by transmission electron microscopy. Electron micrographs were used to measure (F) the percentage of cells with VPs and (G) the diameter of VPs in each condition. ***: p<0.001; NS: not significant (unpaired t-test).

A model for IGF2BP2 involvement in ZIKV life cycle.

Step 1: After NS protein synthesis early after virus entry, IGF2BP2 associates with NS5 and vRNA, thus excluding PUM2 and TNRC6A mRNA from the RNP. Step 2: The infection-induced association between IGF2BP2 RNP and ATL2 allows the targeting of vRNA/NS5 to the ER. Step 3: Viral factors and ATL2 induce the bending of the ER membrane and the formation of VPs allowing highly processive vRNA synthesis. Step 4: IGF2BP2 might be involved in the packaging of vRNA into assembling viruses by targeting the genome to the VP pore. IGF2BP2 recruitment to the replication compartment by be dependent on its mTORC1-dependent phosphorylation status.

IGF2BP2 relocalizes to the viral replication compartment in ZIKV-infected cells at one day post-infection.

Huh7.5 cells were infected with ZIKV H/PF/2013 with a MOI of 10 or left uninfected. At one day post-infection, cells were fixed, immunolabelled for the indicated factors, and imaged by confocal microscopy. Scale bar=10 µm. The white squares indicate the magnified areas in the insets.

HA-tagged IGF2BP2 relocalizes to the viral replication compartment in ZIKV-infected cells, as endogenous IGF2BP2.

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

IGF2BP2 partially co-localizes with NS3 and vRNA in ZIKV infected cells.

FISH with IGF2BP2 and NS3 co-immunostaining was performed in Huh7.5 cells after 2 days post infection with ZIKV at an MOI of 10. White arrows show the triple colocalization. Scale bar=10 µm. The white squares indicate the magnified areas in the insets.

IGF2BP1, IGF2BP3 and YBX1 relocalize to the viral replication compartment in ZIKV-infected cells.

Huh7.5 cells were infected with ZIKV H/PF/2013 with a MOI of 10 or left uninfected. Two days post-infection, cells were fixed, immunolabelled for the indicated factors, and imaged by confocal microscopy. Scale bar=10 µm. The Manders’ coefficients (mean ± SEM) representing the fraction of dsRNA (cyan) and NS3 (red) signals overlapping with IGF2BP3 (A), IGF2BP1 (B) or YBX1 (C) signals are shown (n=number of cells). The white squares indicate the magnified areas in the insets.

LARP1 and DDX5 do not relocalize to the viral replication compartment in ZIKV-infected cells.

(A-B) Huh7.5 cells were infected with ZIKV H/PF/2013 with a MOI of 10 or left uninfected. Two days post-infection, cells were fixed, immunolabelled for the indicated factors, and imaged by confocal microscopy. Scale bar=10 µm. The Manders’ coefficients (mean ± SEM) representing the fraction of dsRNA (cyan) and NS3 (red) signals overlapping with LARP1 (A) or DDX5 (B) signals are shown (n=number of cells). The white squares indicate the magnified areas in the insets. (C-D) The Manders’ coefficients per cell determined from Figures 4B, S4A-C and S5A-B are plotted. Means ± SEM are shown in black.

The association between IGF2BP2 and ZIKV NS5 is RNA-dependent in infected cells.

(A) Huh7.5 cells stably expressing IGF2BP2-HA (+) and control cells (-) were infected with ZIKV H/PF/2013 at a MOI of 10 or left uninfected. Two days later, cell extracts were prepared and subjected to RNase A treatment (+) or not (-) before anti-HA immunoprecipitations. The resulting complexes were analyzed by western blotting for their abundance in the indicated proteins. (B) The RNA content in cell extracts was analyzed on an agarose gel for controlling the efficiency of the RNase A treatment. (C) ZIKV NS5 levels in the IP samples were quantified and means of protein signals (normalized to IGF2BP2) ± SEM based on three independent experiments are shown. ***: p<0.001 (unpaired t-test).

Orthoflavivirus infection modulates the interactome of IGF2BP2.

(A) Heatmap of z-scored protein intensities of all significantly regulated interacting-proteins (ANOVA, FDR < 0.05) across stimuli (mock, DENV-infected, ZIKV-infected) and baits (NT, IGFBP2). (B) Absolute intensity-based quantification (iBAQs) of protein abundance of differential interacting proteins of IGF2BP2 upon DENV or ZIKV infection (N.I.=not identified). (C) Huh7.5 cells expressing IGF2BP2-HA and control cells were infected with ZIKV H/PF/2013 at a MOI of 10 or left uninfected. Two days later, cell extracts were prepared and subjected to anti-HA immunoprecipitations. The resulting complexes were analyzed by western blotting for their abundance in the indicated proteins. (D) ATL2 levels were quantified and means of protein signals (normalized to actin (extracts) and IGF2BP2 (IP)) ± SEM are shown. *: p<0.05; ns: not significant (unpaired t-test).