Fig 1.
Kinetics of the subcellular localization of ORF2 protein.
PLC3 cells electroporated with HEV-p6 (A), HEV-Sar55 (B) and HEV83-2 (C) RNA and Huh-7.5 cells infected with HEV-p6 (D) were fixed at the indicated timepoints post-electroporation (p.e.) (A-C) or post-infection (p.i.) (D). Indirect immunofluorescence analysis was performed using the 1E6 anti-ORF2 antibody. Cells were analyzed by confocal microscopy (magnification x40). Scale bar, 20 μm. Nuclear/cytosolic fluorescence intensity quantification was done using ImageJ software (mean ± S.D., n ≥ 30 cells, Friedman with Nemenyi test). *p < 0.05, ****p < 0.0001. Mock electroporated PLC3 cells (PLC3), cells electroporated with a replication-deficient HEV83-2 strain (PLC3/HEV83-2-GAD) and non-infected Huh-7.5 cells were used as controls.
Fig 2.
ORF2 contains an Arginine-Rich Motif (ARM) that is important for its nuclear localization.
(A) Schematic sequence alignment of HEV-p6 ORF2wt and ARM/SP mutants. (B) Subcellular localization of HEV-p6 ORF2wt and ARM/SP mutants. PLC3 cells were electroporated with wt and mutant HEV-p6 RNAs. At 18 h.p.e, cells were processed for indirect immunofluorescence using the 1E6 anti-ORF2 antibody (Ab) and analyzed by confocal microscopy (magnification x63). Red = ORF2; Blue = DAPI. Scale bar, 20μm. 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 < 0.0001. (C) Subcellular fractionation of PLC3/HEV-p6 expressing ORF2wt and ARM/SP mutants at 10 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. ORF3 protein in cell lysates was detected with a rabbit anti-ORF3 Ab. 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). (D) Infectious titer determination and HEV RNA quantification in PLC3/HEV-p6 expressing ORF2wt or mutant proteins. Extra- and intracellular viral particles were extracted at 10 d.p.e and used to infect naïve Huh-7.5 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 (n = 4). Extra- and intracellular viral RNAs were quantified at 10 d.p.e by RT-qPCR (n ≥ 5) (mean ± S.D., Kruskal-Wallis with Conover’s test). *p < 0.05, **p < 0.01, ****p < 0.0001. (E) PLC3/HEV-p6-wt, PLC3/HEV-p6-5R/5A, PLC3/HEV-p6-PSG/3R and PLC3 mock cells were processed for proximity ligation assay using antibodies to ORF2 and Importin-α1 at 18 h.p.e. Stacks of images corresponding to the total volume of the cells were acquired, and maximum intensity projections of the stacks were generated. For each condition, 12 fields of cells were analyzed (total cell number ≥ 165). Scaled regions of interest of a representative field (left) and quantification of spot/cell (right) are shown (mean ± S.D., Kruskal-Wallis with Conover’s test). ****p < 0.0001.
Fig 3.
Nuclear export of the ORF2 protein.
(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.
Fig 4.
Translocation and maturation of the glycosylated ORF2 forms.
(A) Dose-response inhibition of ORF2g/c secretion in mycolactone-treated cells. At 7 d.p.e., PLC3/HEV-p6 and mock cells were treated for 24h with the indicated concentrations of mycolactone (in nM) or maximal volume of the vehicle, ethanol (indicated as 0 nM). Supernatants (SN) and lysates (Cells) were collected and ORF2 proteins were detected by WB using the 1E6 Ab. Tubulin served as control protein loading. (B) Schematic representation of ORF2i/g/c proteins and recognition sites of P1H1 and P3H2 antibodies used to discriminate the different ORF2 forms. SP, signal peptide. PC, proprotein convertase. Glycans are in black. (C) Immunoprecipitation of ORF2 proteins in SN and lysates of PLC3/HEV-p6 cells by P1H1, P3H2 and isotype control (CTL) antibodies immobilized on magnetic beads. ORF2 proteins were detected by WB using the 1E6 Ab. (D-F) At 7 d.p.e., PLC3/HEV-p6 cells were treated with the indicated concentrations of Decanoyl-RVKR-chloromethylketone (CMK), hexa-D-arginine amide (D6R) and SSM3 trifluoroacetate (SSM3) (in μM) or DMSO diluent (indicated as 0 μM). (G) At 7 d.p.e., PLC3/HEV-p6 and PLC3 mock cells were transfected with siRNA targeting furin (siFur) or non-targeting siRNA (siCTL) or left non-transfected (NT). (D-G) At 72h post-treatment or post-transfection, supernatants (SN) and lysates (Cells) were collected. SN were immunoprecipitated with P1H1 and P3H2 antibodies and ORF2 proteins were detected by WB using the 1E6 Ab. ORF2intra, αV-Integrin (IntαV) and Tubulin (Tub) were detected in cell lysates. In G, furin (Fur) was also detected in cell lysates. αV-pro-integrin (ProintαV) corresponds to the non-maturated αV-integrin. ORF2g* corresponds to the ORF2g immunoprecipitated by the P1H1 Ab. Molecular mass markers are indicated on the right (kDa).
Fig 5.
The ORF2 ARM is the regulator of ORF2 addressing.
Schematic representation of ORF2wt and CD4wt proteins. ORF2 sequences are in blue. ARM residues are highlighted in red. CD4 sequences are in green. (A) H7-T7-IZ cells were transfected with pTM plasmids expressing wt, mutant or chimeric ORF2 proteins. Twenty-four hours post-transfection, cells were fixed and processed for ORF2 staining (in red). Nuclei are in blue. Representative confocal images are shown together with ORF2/DAPI merge images (magnification x63). Blue dots observed in some pictures are DAPI-stained transfected plasmids. A schematic representation of each construct is shown on the left. Scale bar, 20μm. (B) Nuclear-to-cytoplasmic ORF2 staining ratio in H7-T7-IZ cells expressing mutant and chimeric ORF2 proteins. Quantification was done using ImageJ software (mean ± S.D., n ≥ 30 cells, Kruskal-Wallis with Conover’s test). **p < 0.01, ****p < 0.0001. (C) Subcellular fractionation of H7-T7-IZ cells expressing mutant and chimeric ORF2 proteins at 24h post-transfection. Fractionation was done using a subcellular protein fractionation kit for cultured cells. ORF2 proteins were detected by WB with 1E6 Ab. Tubulin, Calnexin (CNX) and Lamin B1 were also detected to control the quality of fractionation. Molecular mass markers are indicated on the right (kDa).
Fig 6.
CD4 addressing by the ORF2 ARM.
Schematic representation of ORF2wt and CD4wt proteins. SP ORF2 residues are shown in blue. ARM residues are highlighted in red. CD4 sequences are in green. (A) and (C), H7-T7-IZ cells were transfected with pTM plasmids expressing CD4wt or chimeric C4 and C5 proteins. Twenty-four hours post-transfection, cells were fixed and processed for CD4 staining (in green). Nuclei are in blue. Representative confocal images are shown together with CD4/DAPI merge images. Blue dots observed in some pictures are DAPI-stained transfected plasmids. A schematic representation of each construct is shown on the left. Scale bar, 20μm. (B) Nuclear-to-cytoplasmic CD4 staining ratio in H7-T7-IZ cells expressing CD4wt or chimeric proteins. Quantification was done using ImageJ software (mean ± S.D., n ≥ 30 cells, Kruskal-Wallis with Conover’s test). ****p < 0.0001. (D) Subcellular fractionation of H7-T7-IZ cells expressing CD4wt or chimeric proteins at 24h post-transfection. Fractionation was done using the subcellular protein fractionation kit for cultured cells. CD4 proteins were detected by WB with a rabbit polyclonal anti-CD4 Ab. Tubulin, Calnexin (CNX) and Lamin B1 were also detected to control the quality of fractionation. Molecular mass markers are indicated on the right (kDa).
Fig 7.
The ORF2 ARM regulates the topology of ORF2 SP.
A schematic representation of differential permeabilization process with Triton X-100 and Digitonin is shown. Representative images of the differential detection of two epitopes on the ER-membrane associated Calnexin (CNX) used to assess the permeabilization conditions are shown. H7-T7-IZ cells were transfected with pTM plasmids expressing wt, mutant or chimeric ORF2 proteins. Twenty-four hours post-transfection, cells were fixed, permeabilized with either Triton X-100 or Digitonin, and processed for ORF2 staining (in red). Nuclei are in blue. Representative confocal merge ORF2/DAPI images are shown (magnification x63). Blue dots observed in some pictures are DAPI-stained transfected plasmids. A schematic representation of each construct is shown on the left and its predicted topology on the right. Blue and red asterisks correspond to 5R/5A and PSG/3R mutations, respectively. Scale bar, 20μm.
Fig 8.
The ORF2 ARM is also able to control the topology of CD4 SP.
A schematic representation of differential permeabilization process with Triton X-100 and Digitonin is shown. Representative images of the differential detection of two epitopes on the ER-membrane associated Calnexin (CNX) in the used permeabilization conditions are shown. H7-T7-IZ cells were transfected with pTM plasmids expressing CD4wt or chimeric C4 and C5 proteins. Twenty-four hours post-transfection, cells were fixed, permeabilized with either Triton X-100 or Digitonin, and processed for CD4 staining (in green). Nuclei are in blue. Representative confocal CD4/DAPI merge images are shown. Blue dots observed in some pictures are DAPI-stained transfected plasmids. A schematic representation of each construct is shown on the left and its predicted topology on the right. Blue and red asterisks correspond to 5R/5A and PSG/3R mutations, respectively. Scale bar, 20μm.
Fig 9.
Model of ORF2 addressing regulation by ARM.
(A) The signal recognition particle (SRP) recognizes the hydrophobic signal peptide (SP) of the ORF2 nascent chain as it emerges from a translating ribosome. The ribosome-nascent chain-SRP complex is targeted to the membrane and interacts with the SRP receptor, resulting in the release of the SP and docking of the ribosome–nascent chain complex to the Sec61 translocon. The ORF2 SP initially inserts head-on in an Nexo/Ccyt orientation, then inverts its orientation to Ncyt/Cexo. (B) The C-terminal end of SP is exposed to ER lumen and is cleaved by signal peptidase, generating a new N-terminus. Translation then resumes, and the nascent ORF2 protein is translocated into the ER lumen where it is glycosylated and likely undergoes maturation by the proprotein convertase furin. This pathway generates the ORF2g/c forms. (C) For a fraction of ORF2 nascent polypeptide chains, the ARM leads the ORF2 SP to retain its Nexo/Ccyt orientation and integrates as reverse signal-anchor, according to the positive-inside rule. The ORF2 protein anchored to the cytosolic side of membrane is likely processed by an intramembrane protease to generate the ORF2i/ORF2 intra protein that is translocated into the nucleus and assembles into viral particles.
Table 1.
Primary antibodies used for Western-blot, immunoprecipitation and indirect immunofluorescence experiments.