HEV ORF2i protein colocalizes and interacts with the AP-1 adaptor complex

We have recently shown that the ERC is hijacked by HEV to serve as a viral factory [9]. Here, we attempted to identify the mechanisms underlying ORF2i localization to the ERC by analyzing the importance of the AP-1 complex in this process. First, PLC3 cells were electroporated with the infectious gt3 p6 strain RNA (PLC3/HEV) (Fig. 1a) or mock electroporated (PLC3) (Fig. S1), and the colocalization between HEV ORF2i protein (using P1H1 mAb that does not recognize the ORF2g/c forms) and the AP-1 adaptor complex was studied by confocal microscopy at 6 days post-electroporation (d.p.e). We analyzed the overlap of fluorescence intensities of ORF2i and AP-1 staining (Fig. 1a and Fig. S1) and calculated Manders’ overlap coefficients (MOC) of ORF2i protein staining in the AP-1 complex staining (Fig. 1f). As previously observed [9, 13], a nugget-like pattern was detected for the ORF2i protein in the perinuclear region. A perinuclear and dot-like staining was observed for the AP-1 complex (Fig. 1a and Fig. S1) which is in accordance with its TGN and endosomal localization. Interestingly, ORF2i protein and AP-1 complex staining highly overlapped in perinuclear regions (Fig. 1a, right panel), with a MOC value of 0.93 (Fig. 1f), indicating that both proteins likely colocalize in perinuclear regions of electroporated PLC3 cells. Importantly, confocal analyses of Huh-7.5 cells electroporated with p6-HEV RNA (Huh-7.5/HEV, Fig. 1b) or infected with HEV particles (Fig. 1c) also showed a colocalization of ORF2i protein and AP-1 complex in perinuclear regions with MOC values of 0.83 and 0.80, respectively (Fig. 1f). Super-resolution microscopy analysis also revealed a significant overlap of ORF2i protein/AP-1 complex fluorescence intensities in these contexts (Fig. 1b-c, right panels). These results indicate that ORF2i and AP-1 colocalization is not cell type-dependent and does not correspond to an artifact of electroporation.

Fig. 1

HEV-gt1 and -gt3 ORF2i protein colocalizes with AP-1 complex in hepatoma cell lines. Electroporated PLC3/HEV (a) or Huh-7.5/HEV (b) cells were fixed at 6 d.p.e, while infected Huh-7.5 cells (c) were fixed at 12 d.p.i. The pTM-ORF2-p6 (d) and pTM-ORF2-Sar55 (e) transfected H7-T7-IZ cells were fixed at 24 h.p.t. Cells were then permeabilized with cold methanol and 0.5% Triton X-100 and double-stained with anti-ORF2i P1H1 and anti-AP-1γ1 antibodies. Staining were analyzed by confocal microscopy. Red = ORF2i; Green = AP-1; Blue = DAPI. Scale bar, 20 µM. (a-e) On the right, line graphs show overlaps of fluorescence intensities of ORF2i and AP-1 staining measured every 50 nm across the region of interest (ROI) highlighted by the white line on the “Merge” micrograph of each panel. (f) Mander’s overlap coefficients (MOC) of the ORF2i labelling in the AP-1 labelling using the whole cell as ROI. Each data dot in the bar chart represents a cell field, and the total number of ORF2-positive cells used to calculate the MOC is indicated. Displayed results come from biological replicates. Mann-Whitney test, ***p < 0.001, ****p < 0.0001

To strengthen our observations, we extended our study to HEV-gt1 by transfecting constructs expressing either HEV-gt1 ORF2 (Sar55 strain) or HEV-gt3 ORF2 (p6 strain) proteins in Huh-7 cells stably expressing the T7 RNA-polymerase (H7-T7-IZ cells) [33]. In this context, gt1 and gt3 ORF2i proteins display a similar subcellular localization pattern, as previously described [13]. They were characterized by a nugget-like perinuclear accumulation and peripheral distribution (Fig. 1d and e). Strikingly, both gt1 and gt3 ORF2i proteins highly colocalized with AP-1 complex in perinuclear regions with MOC values of 0.76 and 0.77 (Fig. 1f), respectively. In addition, as observed in electroporated/infected cells, peaks of fluorescence intensities of both proteins overlapped perfectly (Fig. 1d-e, right panels), indicating that the colocalization between ORF2i protein and AP-1 complex is conserved among HEV-gt1 and -gt3 strains.

AP-1γ1 adaptin silencing alters ORF2i protein localization in viral factories and inhibits viral replication and infectivity

We next evaluated the importance of the AP-1 complex in the HEV lifecycle by transfecting PLC3/HEV cells with small interfering RNA (siRNA) targeting the γ1 subunit of the AP-1 complex (siAP-1γ1) or non-targeting siRNA (siCTL) (Figs. 2 and 3). We silenced the AP-1γ1 subunit because many viruses, including Coronaviruses [24], subvert this adaptin to promote viral infectivity.

Fig. 2

AP-1γ1 silencing affects ORF2i subcellular localization and colocalization with various viral/host cell markers. (a-d) At 6 d.p.e., PLC3/HEV cells were transfected with siRNA targeting AP-1γ1 (siAP-1γ1), with a non-targeting control siRNA (siCTL). At 3 d.p.t, cells were permeabilized with cold methanol and 0.5% Triton X-100 and double-stained with the indicated antibodies. For each double staining, MOC of the ORF2i labelling in the cellular marker/ORF3 labelling and MOC of the STX6 labelling in the TGN46 labelling were determined using the whole cell as ROI. Each data dot in the bar chart represents a cell field, and the total number of ORF2-positive cells used to calculate the MOC is indicated. Displayed results come from biological replicates. Mann-Whitney test, ****p < 0.0001

Fig. 3

AP-1γ1 silencing affects viral RNA secretion and particle production. (a) Supernatants and cell lysates of non-transfected PLC3/HEV (PLC3/HEV/NT), PLC3/HEV/siCTL, PLC3/HEV/siAP-1γ1 or PLC3 cells were generated 3 days after siRNA transfection. In supernatants, ORF2i and ORF2g/c proteins were immunoprecipitated using anti-ORF2i P1H1 or anti-ORF2i/g/c P3H2 antibodies, respectively. An irrelevant mouse IgG antibody was used as an isotype control (Iso). ORF2 proteins were detected by WB using the 1E6 antibody. In cell lysates, silencing of AP-1γ1 was controlled by WB using a rabbit anti-AP-1γ1 antibody. ORF2i protein was detected using the 1E6 antibody. GRP78 and Tubulin proteins were detected using a rat anti-GRP78 antibody and a mouse anti-β-Tubulin antibody, respectively. (b) HEV RNA quantification in PLC3/HEV/siCTL, PLC3/HEV/siAP-1γ1, PLC3/HEV/DMSO, PLC3/HEV/Sofosbuvir-20µM or PLC3 cells after 3 days of transfection/treatment. Extracellular and intracellular viral RNAs were quantified by RT-qPCR. Titers were adjusted to 100% for siCTL/DMSO-treated cells. PLC3/HEV/Sofosbuvir-20µM cells were used as a positive control for replication inhibition. Values are from four independent experiments. Mann-Whitney test, ***p < 0.001, ****p < 0.0001. (c) Infectious titer determination in PLC3/HEV/siCTL, PLC3/HEV/siAP-1γ1, PLC3/HEV/DMSO, PLC3/HEV/Sofosbuvir-20µM or PLC3 cells after 3 days of transfection/treatment. Extracellular and intracellular viral particles were 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. Titers were adjusted to 100% for siCTL/DMSO-treated cells. PLC3/HEV/Sofosbuvir-20µM cells were used as a positive control for infectious titers inhibition. Values are from four independent experiments. Mann-Whitney test, **p < 0.01, ****p < 0.0001

We first carried out an extensive immunofluorescence analysis of the impact of AP-1γ1 silencing on HEV ORF2i protein subcellular localization and colocalization with viral/cellular markers, at 3 days post-transfection. Colocalization analyses were performed by using the P1H1 anti-ORF2i antibody and antibodies against markers related to AP-1 complex shuttling (AP-1, M6PR, Clathrin), TGN compartment (TGN46, STX6), ERC (Rab11) and HEV proteins (ORF3) (Fig. 2 and Fig. S2). Colocalization of markers was quantitatively analyzed by calculating the MOC (Fig. 2, right panels).

As expected, the ORF2i protein significantly colocalized with AP-1 complex in siCTL-transfected cells (MOC = 0.80) in perinuclear regions (Fig. 2a), whereas the ORF2i/AP-1 colocalization was abolished in siAP-1γ1-transfected cells (siAP-1γ1, MOC = 0.06). Interestingly, in AP-1γ1-knocked down cells, ORF2i protein displayed a more diffuse subcellular distribution as compared to siCTL-cells (Fig. 2a). Of note, we found that the siAP-1γ1 transfection had no impact on the subcellular localization of the AP-2 adaptor complex (Fig. S3a), which is involved in cargo endocytosis from plasma membrane to endosomes [15]. These results suggest that the AP-1 complex plays an important role in the subcellular addressing of ORF2i protein.

Next, we analyzed the colocalization between ORF2i protein and the cation-independent mannose-6-phosphate receptor (M6PR), a TGN resident protein that transits between TGN and endosomes [34, 35] (Fig. 2b). The AP-1 complex is known to bind and pack M6PR into transport vesicles at the TGN to deliver it as well as bound lysosomal enzymes to early endosomes, en route to late endosomes and lysosomes [36, 37]. When AP-1-dependent trafficking is disrupted, lysosomal enzymes are secreted instead of being addressed to lysosomes [38]. In PLC3/HEV cells transfected with siCTL, M6PR was detected in perinuclear regions, consistent with its TGN localization, and moderately colocalized with ORF2i protein (MOC = 0.44; Fig. 2b). In cells transfected with siAP-1γ1, M6PR displayed a diffuse dot-like staining in the cytosol, indicating an efficient inhibition of its AP-1-dependent shuttling, and a significant reduction of its colocalization with ORF2i protein was observed (MOC = 0.27; Fig. 2b). The ORF2i protein displayed a more diffuse staining in siAP-1γ1 cells where M6PR displayed a dot-like pattern, suggesting that silencing of AP-1 complex is likely responsible for altering its protein subcellular localization in PLC3/HEV cells.

To strengthen our observations, we examined the impact of AP-1 silencing on TGN compartment integrity to make certain that ORF2i subcellular localization modification was not due to a TGN morphological alteration in siRNA-transfected cells. For that purpose, we analyzed the colocalization of ORF2i protein with TGN46, (Fig. 2c) and colocalization of TGN46 with Syntaxin-6 (STX6), which are both markers of the TGN compartment [39, 40] (Fig. 2d). In siCTL and siAP-1γ1-transfected PLC3/HEV cells, TGN46 subcellular localization remained unchanged (Fig. 2c and d). Although weak, the codistribution of ORF2i with TGN46 was decreased significantly in siAP-1γ1 cells compared to siCTL cells (MOC = 0.18 vs. 0.32) (Fig. 2c). Importantly, subcellular localization and colocalization of TGN46 and STX6 remained unchanged upon siRNA transfection (Fig. 2d), indicating that the TGN integrity was not affected upon AP-1 silencing. Together these observations indicate that the AP-1 complex is likely involved in the subcellular trafficking of the HEV ORF2i protein.

Next, as AP-1 complex-dependent shuttling involves clathrin, we analyzed the colocalization between ORF2i protein and clathrin (Fig. 2e). In siCTL cells, a strong colocalization (MOC = 0.82) was observed in focalized perinuclear regions. In siAP-1γ1 cells, this colocalization was significantly reduced (MOC = 0.40) and clathrin-associated staining was more peripheral, indicating that AP-1 silencing also affects clathrin-recruitment to membranes. These results indicate that the ORF2i protein co-localizes with clathrin in an AP-1 complex-dependent manner.

We have previously shown that the ORF2i protein co-distributes in perinuclear nugget-like structures together with the HEV ORF3 protein and Rab11, a marker of ERC [9]. Therefore, we next studied the impact of AP-1 silencing on the colocalization of ORF2i with ORF3 (Fig. 2f) and Rab11 (Fig. 2g). Regarding the ORF2i/ORF3 co-staining, a diffuse cytosolic dot-like distribution of the HEV ORF3 protein was observed upon siAP-1γ1 transfection, as well as a significantly reduced ORF2i/ORF3 colocalization compared to control condition (MOC = 0.22 vs. 0.79; Fig. 2f). This indicates that AP-1 silencing alters the co-distribution of HEV ORF2i/ORF3 proteins. Regarding the ORF2i/Rab11 co-staining, a strong colocalization of both markers was observed in perinuclear nugget-like structures in control cells (MOC = 0.80; Fig. 2g) whereas their colocalization was significantly impaired upon siAP-1γ1 transfection (MOC = 0.37). These results indicate that the silencing of AP-1γ1 adaptin prevents the ORF2i protein to be targeted to ERC, and hence, to viral factories.

Next, we sought to assess the impact of altered ORF2i protein localization in viral factories upon AP-1γ1 on the viral lifecycle. For this purpose, we analyzed by Western blotting (WB) and immunoprecipitation (IP) the intracellular and extracellular ORF2 protein expression in PLC3/HEV cells transfected with siCTL or siAP-1γ1 (Fig. 3a). In WB, we used the 1E6 mAb that recognizes all the ORF2 isoforms. We also controlled the expression levels of tubulin and the stress-overexpressed GRP78 marker (Fig. 3a). In IP, we used P1H1 and P3H2 antibodies to differentially immunoprecipitate ORF2 forms in cell supernatants. Indeed, we have previously shown that the P1H1 mAb specifically immunoprecipitates the particle-associated ORF2i protein whereas the P3H2 mAb preferentially immunoprecipitates glycosylated ORF2g/c forms from heat-denatured HEV-cell culture supernatant [9]. Impact of the AP-1 silencing on viral production was also analyzed by quantification of viral RNAs (Fig. 3b) and infectious titers (Fig. 3c) and compared to cells treated with sofosbuvir (Sof), a well-characterized inhibitor of in vitro HEV replication and production [41].

The silencing of AP-1γ1 adaptin did not induce any cellular stress response (i.e. no impact on intracellular GRP78 detection levels in WB) and did not affect the intracellular expression level/pattern of ORF2 protein (Fig. 3a, Cells). In contrast, we found that the AP-1γ1 silencing induced a significant reduction of ORF2i protein detection in supernatants after immunoprecipitation with the anti-ORF2i P1H1 antibody, without affecting secretion of glycosylated ORF2g/c forms (Fig. 3a, Supernatant). These results suggest that the AP-1γ1 silencing likely reduces the secretion of viral particles. Consistently, the levels of extracellular viral RNA (Fig. 3b) and extracellular infectious viral particles (Fig. 3c) were reduced. Of note, intracellular HEV RNA levels were reduced by 40% upon AP-1γ1 silencing (Fig. 3b), indicating that the adaptor complex might be important for a viral or cellular factor involved in HEV replication. Indeed, the silencing of AP-1γ1 or AP-1γ2 adaptins in PLC3 cells stably replicating a p6 subgenomic replicon led to a 38% and 36% reduction in HEV RNA levels, respectively (Fig. S4), confirming that AP-1 complex plays a role in HEV replication. Strikingly, intracellular infectious titers were reduced by 80% upon AP-1γ1 silencing (Fig. 3c). These results indicate that the inhibition levels of intracellular viral RNA and infectious titers cannot be correlated and suggest that, in addition to an effect on HEV replication, AP-1γ1 silencing likely affects another step of the HEV life cycle, namely the assembly of infectious viral particles.

Taken together, these results highlight the importance of the AP-1 complex in HEV replication and addressing the ORF2i protein to viral factories for producing infectious viral particles. The AP-1 complex is probably a central player in the HEV lifecycle.

Pharmacological inhibition of the AP-1 complex prevents the localization of ORF2i protein in viral factories and inhibits viral infectivity

Since in AP-1 silencing experiments, we observed an effect on ORF2 subcellular localization, but also on HEV replication, we then wanted to use another approach to define the importance of AP-1 in the HEV lifecycle. We therefore used the pharmacological inhibitor A5 that specifically inhibits the AP-1 complex [25, 42, 43]. This compound is a cell-permeable piperazinyl phenylethanone derivate that specifically inhibits AP-1 complex by acting at a step after adaptor membrane recruitment, thereby significantly impairing AP-1 subcellular shuttling [42] (Fig. S5a). Therefore, we next sought to analyze the effect of this AP-1 traffic inhibitor, at non-cytotoxic dose of 150µM (Fig. S5b), on the subcellular localization of the ORF2i protein and the HEV lifecycle.

We first carried out an extensive immunofluorescence analysis of the impact of A5 treatment on HEV ORF2i protein subcellular localization and colocalization with viral/cellular markers, at 3 days post-treatment (Fig. 4), as described above.

Fig. 4

AP-1 complex pharmacological inhibition by A5 impairs ORF2i subcellular localization and colocalization with various viral/host cell markers. (a-g) At 6 d.p.e., PLC3/HEV cells were treated with H2O or A5 (150µM) and fixed after 3 days of treatment. Then, cells were permeabilized with cold methanol and 0.5% Triton X-100 and double-stained with the indicated antibodies. For each double staining, MOC of the ORF2i labelling in the cellular marker/ORF3 labelling was determined using the whole cell as ROI. Each data dot in the bar chart represents a cell field, and the total number of ORF2-positive cells used to calculate the MOC is indicated. Displayed results come from biological replicates. Mann-Whitney test, ****p < 0.0001

As shown in Fig. 4a, the ORF2i protein significantly colocalized with AP-1 complex in H2O-treated cells (MOC = 0.86) in perinuclear regions whereas this colocalization was significantly reduced in A5-treated cells (A5–150µM, MOC = 0.45). Interestingly, in A5-treated cells, ORF2i protein displayed a more diffuse subcellular distribution as compared to H2O-treated cells (Fig. 4a), similarly to what was observed in siAP-1γ1-transfected cells (Fig. 2a). As observed for the siAP-1γ1 transfection (Fig. S3a), we found that treatment with A5 compound had no impact on the subcellular localization of the AP-2 adaptor complex (Fig. S3b), confirming that this compound is specific to AP-1 complex. These results confirm the importance of the AP-1 complex in the subcellular addressing of ORF2i protein.

In line with silencing experiments, the M6PR marker also displayed a more diffuse cytosolic pattern and a reduced co-distribution with ORF2i (MOC = 0.49 in H2O-cells vs. 0.22 in A5-cells; Fig. 4b) upon treatment with A5, without any impact on TGN integrity (Fig. 4c-d), indicating that the A5 treatment alters AP-1 complex activity.

Clathrin also showed a more diffuse signal in the cytosol and reduced colocalization with ORF2i in A5-treated cells (MOC = 0.88 in H2O-cells vs. 0.50 in A5-cells; Fig. 4e). This indicates that AP-1 complex activity as well as clathrin recruitment are important for the subcellular addressing of ORF2i protein. Regarding ORF2i/ORF3 co-staining (Fig. 4f), ORF3 staining was less focalized in treated cells, with a significantly reduced MOC value in A5-treated cells (MOC = 0.22), as compared to siCTL cells (MOC = 0.80). This indicates that the inhibition of AP-1 complex alters HEV ORF2i/ORF3 proteins co-distribution. For ORF2i/Rab11 co-staining (Fig. 4g), a strong colocalization was observed in perinuclear nugget-like structures in the control condition (H2O, MOC = 0.82) whereas upon treatment with A5, the colocalization was significantly reduced (MOC = 0.42), indicating that pharmacological inhibition of AP-1 complex trafficking prevents the ORF2i protein from being targeted to viral factories. Altogether, these results confirm that AP-1 complex is an important host determinant involved in ORF2i protein addressing to viral factories.

Next, we sought to assess the impact of altered ORF2i protein localization in viral factories upon A5 treatment on the viral lifecycle. For that purpose, we proceeded as in Fig. 3 with PLC3/HEV cells treated with H2O or A5 (Fig. 5). As observed for the siRNA transfected cells, we found that the A5 treatment did not affect intracellular expression levels/patterns of ORF2 and ORF3 proteins (Fig. 5a, Cells). In addition, A5 treatment did not affect intracellular expression level/pattern of AP-1 complex and did not induce any cellular stress response (i.e. no impact on intracellular GRP78 detection levels in WB). Interestingly, we found that A5 treatment induced a significant reduction of ORF2i protein detection in supernatants after immunoprecipitation with the anti-ORF2i P1H1 antibody, without affecting secretion of glycosylated ORF2g/c forms (Fig. 5a, Supernatant). These results indicate that A5 treatment likely reduces the secretion of viral particles. Consistently, the levels of extracellular viral RNA (Fig. 5b) and extracellular infectious viral particles (Fig. 5c) were reduced. In contrast to siAP-1γ1-transfected cells (Fig. 3b), intracellular viral RNA levels were significantly increased upon A5 treatment (Fig. 5b). Since we observed that the treatment of PLC3 cells stably replicating a p6 subgenomic replicon with A5 compound only slightly reduced HEV RNA levels (Fig. S4), we hypothesized that the inhibition of secretion and/or assembly of viral particles by A5 leads to an intracellular accumulation of HEV RNA (Fig. 5b). Consistently, levels of intracellular infectious particles were significantly reduced upon A5 treatment (≈ 70%; Fig. 5c), indicating that A5 inhibits HEV assembly and consequently viral particle secretion. Taken together, these results indicate that inhibition of AP-1 complex activity impairs assembly and therefore secretion of infectious HEV particles.

Fig. 5

AP-1 complex pharmacological inhibition by A5 alters viral RNA secretion and particle production. (a) Supernatants and cell lysates of PLC3/HEV/H2O, PLC3/HEV/A5-150µM, PLC3/H2O cells were generated after 3 days of treatment. In supernatants, ORF2i and ORF2g/c proteins were immunoprecipitated using anti-ORF2i P1H1 or anti-ORF2i/g/c P3H2 antibodies, respectively. An irrelevant mouse IgG antibody was used as an isotype control (Iso). ORF2 proteins were detected by WB using the 1E6 antibody. In cell lysates, ORF2i protein was detected by WB using 1E6 antibody. GRP78 and Tubulin proteins were detected using a rat anti-GRP78 antibody and a mouse anti-β-Tubulin antibody, respectively. (b) HEV RNA quantification in PLC3/HEV/H2O, PLC3/HEV/A5-150µM, PLC3/HEV/DMSO, PLC3/HEV/Sofosbuvir-20µM or PLC3 cells after 3 days of treatment. Extracellular and intracellular viral RNAs were quantified by RT-qPCR. Titers were adjusted to 100% for H2O/DMSO-treated cells. PLC3/HEV/Sofosbuvir-20µM cells were used as a positive control for replication inhibition. Values are from two independent experiments. Mann-Whitney test, **p < 0.01. (c) Infectious titer determination in PLC3/HEV/H2O, PLC3/HEV/A5-150µM, PLC3/HEV/DMSO, PLC3/HEV/Sofosbuvir-20µM or PLC3 cells after 3 days of treatment. Extracellular and intracellular viral particles were 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. Titers were adjusted to 100% for H2O/DMSO-treated cells. PLC3/HEV/Sofosbuvir-20µM cells were used as a positive control for infectious titers inhibition. Values are from three independent experiments. Mann-Whitney test, **p < 0.01, ****p < 0.0001

Taken together, these results confirm the importance of the AP-1 complex in addressing the ORF2i protein to viral factories and thus assembly and secretion of viral particles. The AP-1 complex is therefore a key player in the HEV lifecycle.

AP-1 complex-dependent shuttling is pivotal for the HEV lifecycle in PHHs

To further validate our findings in a cell culture system of hepatocytes closer to in vivo settings, we analyzed the impact of A5 inhibitor in HEV-infected primary human hepatocytes (PHHs) (Fig. 6). Briefly, one day after seeding (D0), PHHs were infected with HEV particles and treated with the compound A5, ribavirin (RBV, an FDA-approved HEV replication inhibitor), or their respective diluent (H2O or DMSO). At 3 days post-infection/treatment, supernatants were recovered (Fig. 6a). We assessed the impact of treatments on ORF2 protein detection in PHH supernatant by WB (Fig. 6b) and on viral production by quantification of infectious titers (Fig. 6c).

Fig. 6

AP-1 complex is involved in particle production in primary human hepatocytes. (a) Experimental procedure. One day after seeding (D0), primary human hepatocytes (PHHs) were infected with intracellular HEV particles and treated with the appropriate drug/diluent. At D + 1 and D + 2, fresh medium containing drug/diluent was added to the wells. At D + 3, supernatants were recovered and processed for WB and extracellular infectious titers determination. D = Day. (b) Supernatant of HEV-infected PHHs or Mock PHHs, treated or not with diluent (H2O or DMSO), A5-150µM (A5) or Ribavirin-25µM (RBV) were subjected to western blotting to detect the ORF2g protein using the 1E6 Ab. The asterisk indicates a non-specific band detected in HEV-infected cells. (c) Supernatants were used to infect naïve Huh-7.5 cells for 3 days to determine extracellular infectious titers. 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 relative infectious titers. PHHs-Ribavirin-25µM (RBV) cells were used as a positive control for infectious titers inhibition. Values are from two independent experiments. Mann-Whitney test, **p < 0.01

The low amount of virus particles produced in the supernatants of infected PHH prevents the detection or immunoprecipitation of the ORF2i form, as carried out in Figs. 3 and 5. However, the ORF2g form, which is the predominant ORF2 form in cell culture supernatants [7, 8], can be easily detected by WB. This form follows the general secretion pathway, which is different from that of ORF2i protein [13]. As shown in Fig. 6b, A5 treatment did not affect the detection level/pattern of the ORF2g protein. In contrast, no ORF2g-associated signal was detected in the RBV-treated condition, due to RBV-induced replication inhibition. These results indicate that the A5 compound has not impact on the secretion of the ORF2g protein in PHH supernatant and, more globally, on the protein secretion of PHHs.

Next, we analyzed the impact of A5 treatment on viral production by determining the extracellular infectious titers (Fig. 6c). After treatment with A5, infectious titers were significantly reduced (≈ 40%) as compared to control conditions, to levels comparable to the RBV-treated condition (≈ 40%). This indicates that A5 treatment inhibits the production of viral particles in HEV-infected PHHs, as observed in hepatoma cell lines.

Taken together, these results indicate that AP-1-dependent shuttling is pivotal for the HEV particle secretion in this ex vivo system, without affecting the secretion of ORF2g protein. These results also confirm our conclusions drawn in hepatoma cell lines underlining the pivotal role of AP-1 complex in the HEV lifecycle.

HEV ORF2i protein interacts with the AP-1 adaptor complex

Next, we studied the interaction between the HEV ORF2i protein and the AP-1 complex (Fig. 7). First, we performed co-immunoprecipitation (co-IP) experiments in PLC3/HEV and PLC3 cells, at 6 d.p.e. (Fig. 7a). AP-1 complex was detected at similar intensities in PLC3/HEV and PLC3 cell lysates whereas ORF2i protein was only detected in PLC3/HEV cell lysate (“Inputs”, Fig. 7a). Following immunoprecipitation of AP-1 complex (IP AP-1), a faint band corresponding to the ORF2i protein was detected in PLC3/HEV cells (arrowhead) and not in PLC3 mock cells or in the isotype control condition (Iso), indicating that the ORF2i protein was specifically co-immunoprecipitated with AP-1 (Fig. 7a). Therefore, the AP-1 complex and the HEV ORF2i protein likely interact specifically in PLC3/HEV cells. Immunoprecipitation with anti-ORF2i P1H1 mAb was performed but failed to detect the AP-1 complex, probably because the P1H1 epitope is hidden by the interaction (data not shown).

Fig. 7

HEV ORF2i protein interacts with AP-1 complex in hepatoma cell lines. (a) PLC3/HEV and PLC3 cell lysates were immunoprecipitated using a polyclonal anti-AP-1antibody (IP AP-1). An irrelevant rabbit IgG antibody was used as an isotype control (Iso). Inputs and immunoprecipitated AP-1 and ORF2 proteins were next detected by WB. (b) PLC3/HEV/H2O, PLC3/HEV/A5-150µM and PLC3/H2O cells were processed for proximity ligation assay using anti-ORF2i P1H1 and anti-AP-1γ1 antibodies after 3 days of treatment. 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 > 150 cells). Single stack of representative fields and quantification of spot/cell (bottom right) are shown. Kruskal-Wallis test, **p < 0.01, ****p < 0.0001. Molecular modeling of the interaction between ORF2i domain S and AP-1 complex subunit σ1. (c) Experimental overall structure of a truncated ORF2 form (aa 112–606) in the context of an icosahedral virus-like particle. The ordered domains S, M and P are colored in green, magenta and gray, respectively. The side chains of leucine 163 and leucine 164 are displayed as cyan spheres in (c) and (d). (d) AlphaFold2 mod