Figure 6.

The residue Gln109 in the KPNA2 CMA motif is indispensable for NS2A-mediated reduction and ZIKV replication increases in the cells with KPNA2 knockdown. (A) Schematic illustration of KPNA2 mutation at the potential CMA motif. The numbers above the lines indicate KPNA2 amino acid residues. The underlined amino acid residues indicate point mutations. M1: mutation 1 (L104A and S105A), M2: R106A and K108A; M3: E107A, and M4: Q109A. (B) Effect of the point mutations in KPNA2 on NS2A-mediated KPNA2 reduction. WT KPNA2 and EV were included as controls. (C) Mutant KPNA2^Q109A has much less interaction with LAMP2A than WT KPNA2. HEK293 cells were transfected with KPNA2-M4 or WT KPNA2 plasmids. The cells were treated with NH₄CL for 6 h before harvested for co-IP. (D) The co-IP precipitate of mutant KPNA2^Q109A contains much less NS2A than the precipitate of WT KPNA2. HEK293 cells were transfected with NS2A-YFP and KPNA2-M4 or WT KPNA2 plasmids. The cells were treated with NH₄CL for 6 h before harvested for co-IP. (E and F). KPNA2 knockdown in Vero cells leads to higher ZIKV replication than control shRNA. Vero cells stably transduced with recombinant retrovirus expressing control shRNA (C-shRNA) or shRNA against KPNA2 (shKPNA2) were infected with ZIKV and harvested 48 hpi. ZIKV titers are shown as Log10/ml on Y-axis. A significant difference from cells with C-shRNA is denoted with *** for P< 0.001. (G) KPNA2 knockdown has minimal effect on Vero cell viability. NS: no significant difference. (H, I and J). KPNA2 knockdown in MARC-145 cells leads to significantly higher ZIKV replication than control shRNA in MARC-145 cells, whereas the cell viability remains stable