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. 2014 Nov 19;89(3):1719–1730. doi: 10.1128/JVI.02639-14

FIG 4.

FIG 4

Reconstitution of hexon binding by expression of the N-terminal domain of Nup214. (A) Expression of Nup214 domains in HeLa cells. HeLa cells were transfected with expression constructs corresponding to the N-terminal domain of Nup214 (aa 1 to 1058) or the coiled-coil domain of Nup214, Nup214(586–1058), or were not transfected (NT). Total lysates were analyzed by immunoblotting using V5 antibody. The predicted sizes of the domains are indicated by the arrows. (B) Representative images of hexon binding at the NE. HeLa cells were transfected with shRNA expression plasmid against Nup214 (sh214-3) alone or were cotransfected with the Nup214 coiled-coil domain expression plasmid [sh214-3/Nup214(586–1058)] or with the Nup214 N-terminal domain expression plasmid [sh214-3/Nup214(1–1058)] or were not transfected (NT). Purified hexon was added to digitonin-permeabilized HeLa cells. The cells were analyzed by IF staining using FITC anti-V5 antibody to detect the overexpressed Nup214 fragments or with the anti-hexon antibody and anti-Nup214 antibody. The white arrows indicate hexon staining around the nucleus of Nup214-depleted cells after overexpression of the N-terminal domain of Nup214(1–1058). Symbols in the schematic representations at left are as identified on Fig. 2A. (C) Quantitative analysis of hexon binding at the NE. The histogram presents the mean fluorescence intensity of hexon staining indicated as a percentage. The mean fluorescence intensity of hexon staining around the nucleus was measured (n = 43 to 91 cells for each condition of each experiment) and compared to that in NT cells, set at 100 (***, P < 0.001). (D) Reconstitution of the nuclear import of protein VII by expression of the N-terminal domain of Nup214 in Nup214-depleted cells. HeLa cells were transfected with expression plasmid eGFP-shRNA against Nup214 (eGFP-sh214-3) alone or were cotransfected with the Nup214 coiled-coil domain expression plasmid [eGFP-sh214-3/Nup214(586–1058)] or with the Nup214 N-terminal domain expression plasmid [eGFP-sh214-3/Nup214(1–1058)] or were not transfected (NT). Cells were infected with AdV for 3 h. The cells were analyzed by IF staining using anti-pVII antibody. The histogram presents the mean fluorescence intensity of pVII staining in the nucleus, indicated as a percentage. The mean fluorescence intensity of pVII staining around the nucleus was measured (n = 40 to 114 cells for each condition of each experiment) and compared to that in NT cells, set at 100 (*, P < 0.05).