Figure 10. IGF2BP2 regulates the biogenesis of Zika virus (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 vesicle packet (VP) induction. Finally, the activity of HDV ribozyme ensures that the 3’ terminus of the RNA is similar to that of viral RNA (vRNA) genome. Huh7-Lunet-T7 were transduced with short-hairpin RNA (shRNA)-expressing lentiviruses at an 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).