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. 2022 Aug 25;18(8):e1010798. doi: 10.1371/journal.ppat.1010798

Fig 3. Nuclear export of the ORF2 protein.

Fig 3

(A) Analysis of ORF2 nuclear export in inhibitors treated-PLC3/HEV-p6 cells. Cells were treated at 32 h.p.e with 20nM of Leptomycin B (LepB), 100nM of Verdinexor (Verd) or diluent (EtOH or DMSO, respectively) for 16h. Cells were processed for indirect immunofluorescence with the 1E6 anti-ORF2 Ab and analyzed by confocal microscopy (magnification x63). Red = ORF2; Blue = DAPI. (B) Schematic representation of HEV-p6 ORF2 protein sequence highlighting the three studied NES motifs (i.e., NES9, NES10 and NES12). (C) Subcellular localization of ORF2 NES mutants at 48 h.p.e. Red = ORF2; Blue = DAPI. In A and C, the scale bars correspond to 20μm, and nuclear/cytosolic fluorescence intensity quantification was done using ImageJ software (mean ± S.D., n ≥ 30 cells, Kruskal-Wallis with Conover’s test). *p < 0.05, ***p < 0.001, ****p < 0.0001. (D) Subcellular fractionation of PLC3/HEV-p6 expressing ORF2wt and NES mutants at 4 d.p.e. Fractionation was done using a subcellular protein fractionation kit for cultured cells. ORF2 proteins were detected by WB with 1E6 Ab. Glycosylated ORF2 (ORF2g), cleaved ORF2 (ORF2c), intracellular ORF2 (ORF2intra), nuclear ORF2intra (ORF2ni), nuclear and cleaved ORF2intra (ORF2nc) are indicated. ORF2 and ORF3 proteins in total cell lysates were detected with 1E6 Ab and a rabbit anti-ORF3 Ab, respectively. Tubulin, ER marker Calnexin (CNX) and the transcription factor SP1 used as a nuclear marker, were also detected to check the quality of fractionation. 2E2 and 4B2 are conformation-specific anti-ORF2 antibodies. Molecular mass markers are indicated on the right (kDa). (E) Infectious titer determination in PLC3/HEV-p6 expressing ORF2wt or NES mutants. Extra- and intracellular viral particles were extracted at 10 d.p.e and used to infect naïve Huh7.5 cells for 3 days. Cells were next processed for indirect immunofluorescence. ORF2-positive cells were counted and each positive cell focus was considered as one FFU. Results were expressed in FFU/ml. (F) HEV RNA quantification in PLC3/HEV-p6 expressing ORF2wt or NES mutants. Extra- and intracellular viral RNAs were quantified at 10 d.p.e by RT-qPCR. In E and F, n = 6, mean ± S.D., Kruskal-Wallis with Conover’s test. ***p < 0.001, ****p < 0.0001.