Targeting cellular cathepsins inhibits hepatitis E virus entry

Abstract

Background and Aims:

HEV is estimated to be responsible for 70,000 deaths annually, yet therapy options remain limited. In the pursuit of effective antiviral therapies, targeting viral entry holds promise and has proven effective for other viruses. However, the precise mechanisms and host factors required during HEV entry remain unclear. Cellular proteases have emerged as host factors required for viral surface protein activation and productive cell entry by many viruses. Hence, we investigated the functional requirement and therapeutic potential of cellular protease during HEV infection.

Approach and Results:

Using our established HEV cell culture model and subgenomic HEV replicons, we found that blocking lysosomal cathepsins (CTS) with small molecule inhibitors impedes HEV infection without affecting replication. Most importantly, the pan-cathepsin inhibitor K11777 suppressed HEV infections with an EC₅₀ of ~0.02 nM. Inhibition by K11777, devoid of notable toxicity in hepatoma cells, was also observed in HepaRG and primary human hepatocytes. Furthermore, through time-of-addition and RNAscope experiments, we confirmed that HEV entry is blocked by inhibition of cathepsins. Cathepsin L (CTSL) knockout cells were less permissive to HEV, suggesting that CTSL is critical for HEV infection. Finally, we observed cleavage of the glycosylated ORF2 protein and virus particles by recombinant CTSL.

Conclusions:

In summary, our study highlights the pivotal role of lysosomal cathepsins, especially CTSL, in the HEV entry process. The profound anti-HEV efficacy of the pan-cathepsin inhibitor K11777, especially with its notable safety profile in primary cells, further underscores its potential as a therapeutic candidate.

INTRODUCTION

HEV is the main cause of acute hepatitis worldwide, affecting at least 20 million people annually, leading to 3.3 million symptomatic infections and approximately 70,000 fatalities.¹ While HEV infections often remain asymptomatic, they can progress to chronic disease in patients who are immunocompromised, notably transplant recipients and patients with cancer, resulting in rapid progression to cirrhosis and liver failure.² Current therapeutic options against HEV are limited to the off-label use of the broad-spectrum antiviral agent ribavirin (RBV) and pegylated interferon-alpha.³ However, RBV therapy is often discontinued due to adverse effects and is only effective in ∼80% of patients, while 20% of treated patients remain viremic.^3,4

HEV is a member of the Hepeviridae family and has a 7.2 kb single-stranded RNA genome with positive polarity.^5,6 The genome of HEV encompasses 3 open reading frames (ORFs), which encode for a nonstructural polyprotein (ORF1) essential for viral replication,⁷ the capsid protein (ORF2), which undergoes endoplasmic recycling^8–10 and a small ORF3 protein with multiregulatory functions.^11–13 HEV is a quasi-enveloped virus; it exists either as a nonenveloped form (neHEV) in bile or circulates as a host cell lipid-derived enveloped form (eHEV) in the blood.¹⁴ Historically, efficient in vitro culture systems have been lacking, leaving many facets of the HEV life cycle, including virus-host interactions that facilitate attachment to cells and/or entry largely elusive.¹⁵ However, current evidence suggests distinct entry mechanisms for both HEV forms.¹⁶ Notably, studies have shown that neHEV-like particles adhere to cells by means of heparan sulfate proteoglycans.¹⁷ Others have demonstrated that neHEV uses the transmembrane protein integrin α3 and eHEV, the T cell Ig mucin domain 1 receptor for host cell entry.^18,19 Recently, the receptor tyrosine kinase EGF receptor has been identified as a crucial co-factor for both neHEV and eHEV entry.²⁰ Both HEV forms are internalized through a clathrin-dependent and dynamin-2-dependent, receptor-mediated endocytosis.^16,21,22 For eHEV, entry was suggested to involve trafficking to late endosomes and to be dependent on small GTPases Rab5 and Rab7, endosomal acidification, and lysosome-mediated lipid degradation.¹⁶ Yet, the uncoating process, especially for neHEV, remains poorly understood.

Given the pivotal role of viral surface attachment to and entry into host cells in dictating viral host range, tissue tropism, and pathogenesis,²³ viral entry inhibitors offer a rational basis for the development of potent antivirals. In this study, the requirement for cellular proteases during neHEV and eHEV infection on viral entry was examined. Our results suggest that cysteine proteases of the cathepsin (CTS) family, in particular cathepsin L (CTSL), are critical host factors required during both neHEV and eHEV entry and provide a promising target to impede virus uptake into permissive cells.

METHODS

Eukaryotic cell culture

Hepatoma cell lines Huh7.5 and HepG2 were grown in DMEM (Gibco) supplemented with 10% fetal calf serum (GE Healthcare), 100 µg/mL of streptomycin, 100 IU/mL of penicillin (Gibco), 2 mM L-glutamine and 1% nonessential amino acids (Gibco) (DMEM complete) at 37°C in a 5% (v/v) CO₂ incubator. Hepatoma cell line HepG2/C3A cells were grown in Minimum Essential Medium (Gibco) supplemented with 10% ultra-low IgG fetal calf serum (GE Healthcare), 100 μg/mL of gentamycin, 1% sodium pyruvate (Gibco), 2 mM L-glutamine (Gibco) and 1% nonessential amino acids (Gibco) (Minimum Essential Medium complete). The human hepatoma cell line S10-3 (a kind gift from Suzanne Emerson, National Institutes of Health) was cultured in DMEM+GlutaMAX-I supplemented with 10% fetal bovine serum (Capricorn), 1% penicillin/streptomycin (Gibco). Commercially obtained human CTSL (Cathepsin L/MEP) knockout HEK-293T cells (abcam; ab266521) and human wild-type HEK293T cells (abcam; ab255449) were maintained in DMEM complete. The HepaRG cell line was passaged and maintained in William’s E (Gibco) supplemented with 10% fetal calf serum (Gibco), 100 µg/mL Streptomycin (Gibco), 100 U/mL Penicillin (Gibco), 2 mM GlutaMax (Gibco), 5 µg/mL insulin (Sigma-Aldrich, Taufkirchen, Germany), and 50 µM hydrocortisone hemisuccinate (Sigma-Aldrich) (HepaRG medium). For differentiation into mature hepatocytes, cells were seeded at a density of 50,000 cells/well of a 24-well plate and grown for at least 2 weeks until forming confluent layers. The medium was exchanged twice a week. Upon forming confluent layers, differentiation was triggered by adding 1.8% Hybri-Max DMSO (Sigma-Aldrich) to the HepaRG medium. Cells were passaged for at least 2 more weeks in the presence of 1.8% DMSO with 2 medium exchanges per week before performing experiments. Successful differentiation into cholangiocyte-like and hepatocyte-like cells was validated by immunostaining against albumin (Agilent, Cat. Nr. A0001). Primary human hepatocytes (PHHs) were purchased from Primacyt (Schwerin, Germany) as cryopreserved hepatocytes and thawed according to the manufacturer’s instructions (Supplemental Table S1, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). PHHs were seeded on 24-well plates and kept in Human Hepatocyte Maintenance Medium (Primacyt). Donors were serologically tested negative for the following infectious diseases: HIV, hepatitis B and C, and SARS-CoV-2. Patient informed consent was obtained by Primacyt, as stated on their website.

All materials and methods describing assays used in this study are specified in the Supplemental Information, Supplemental Digital Content 1, http://links.lww.com/HEP/I446.

RESULTS

The HCV NS3/4A protease inhibitor telaprevir suppresses HEV infection in human liver cells

During co-infection studies with HCV and HEV,²⁴ we noticed that the HCV NS3/4A serine protease inhibitor telaprevir (TLV), not only blocked HCV replication but also inhibited HEV infection. To further investigate this unexpected effect, we evaluated the anti-HEV activity of TLV against HEV infection in a recently established HEV cell culture model²⁵ (Figure 1). Human hepatoma cells were infected with nonenveloped cell culture-derived HEV (neHEV_CC) in the presence of either TLV or vehicle control (DMSO). TLV effectively inhibited neHEV_CC in Huh7.5 cells (Figure 1A, B) in a concentration-dependent manner with a half-maximum EC₅₀ of 1.5 µM (Figure 1C). Notably, cell viability was marginally reduced (Figure 1A, C). To specifically investigate the potential impact of TLV on HEV replication, we transfected subgenomic HEV-3 reporter replicon (Kernow-C1/p6-Gaussia luciferase) into hepatoma cells and treated cells with 10 µM TLV. We found that HEV replication was not affected by TLV treatment (Figure 1D), suggesting that TLV, though originally developed as an HCV replication inhibitor, influences the establishment of HEV infection rather than HEV RNA replication.

FIGURE 1.

Effect of HCV NS3/4A serine protease inhibitor TLV on HEV infection and replication. (A) Huh7.5 hepatoma cells were inoculated with HEV in the presence of either 10 µM TLV or DMSO (VC) for 4 days before fixation, immunofluorescence staining, and analysis. Statistical significance was determined by 2-tailed paired t test. p-values >0.05 were considered to be not significant (ns). (B) Representative immunofluorescence images illustrate the impact of TLV on the infectivity of nonenveloped cell culture-derived HEV. ORF2=green; DAPI=blue; scale bar=200 µm. (C) Dose-dependent nonenveloped cell culture-derived HEV inhibition by TLV. Normalized infections in percent (%) were fitted by 4-parameter log-logistic model to determine EC₅₀. (D) Replication capacity of Kernow-C1/p6 strain subgenomic replicon during treatment with TLV. Depicted are normalized RLU measured 4, 24, 48, and 72 hours h p.e. RBV and DMSO were employed as positive and negative controls, respectively. The depicted values represent means+SD (A, D) or ±SD (C) from 3 independent experiments. Abbreviations: h p.e., hours post-electroporation; ORF, open reading frame; RBV, ribavirin; RLU, relative light units; TLV, telaprevir; VC, vehicle control.

Leupeptin, a cysteine and serine protease inhibitor, suppresses HEV infection

Given that TLV is an HCV protease inhibitor, but HEV lacks viral proteins with known protease activities, we hypothesized that HCV protease inhibitors might target cellular-encoded proteases essential for HEV infection. To test this, we evaluated the effects of different broad-spectrum protease inhibitors (aprotinin, pepstatin, and leupeptin) on HEV infection through dose-response assays (Figure 2A). Notably, neither aprotinin, a serine protease inhibitor (EC₅₀ > 10 µM), nor pepstatin, an aspartic protease inhibitor (EC₅₀ > 10 µM), reduced HEV infectivity (Figure 2B, C, E). In contrast, the cysteine and serine protease inhibitor leupeptin displayed a concentration-dependent inhibitory effect on HEV infectivity (EC₅₀=1.7 µM) (Figure 2D, E), implying a crucial role of cysteine proteases during HEV infection. Importantly, all tested protease inhibitors were noncytotoxic (EC₅₀ > 10 µM for aprotinin, pepstatin, and leupeptin) (Figure 2E). To specifically investigate the potential impact of the different protease inhibitors on HEV replication, we transfected hepatoma cells with subgenomic HEV-3 reporter replicon (Kernow-C1/p6-Gaussia luciferase) and treated with 10 µM of protease inhibitors. None of the protease inhibitors reduced HEV replication (Figure 2F), further indicating that cellular cysteine proteases are involved in the establishment of HEV infection but not HEV RNA replication.

FIGURE 2.

Role of cellular proteases in HEV infection. (A) Respective specificity of various broad-spectrum protease inhibitors. (B–D) Hepatoma HepG2/C3A cells were infected with nonenveloped cell culture-derived HEV and incubated with protease inhibitors or vehicle control (VC) for 4 days until immunofluorescence staining was performed against the HEV ORF2 capsid protein. Dose-dependent effects of aprotinin (B), pepstatin (C), and leupeptin (D) on nonenveloped cell culture-derived HEV infections were plotted and fitted by 4-parameter log-logistic model. (E) EC₅₀ and CC₅₀ values of aprotinin, pepstatin, and leupeptin in HepG2/C3A cells against infectious HEV Kernow-C1/p6 virus, determined by dose-response curves. To test the significance of mean differences, one-way ANOVA, followed by Dunnett multiple comparison test, was used. p-values >0.05 were considered to be not significant (ns). (F) Impact of protease inhibitors on the replication of HEV subgenomic reporter replicon based on the Kernow-C1/p6 strain. Depicted are normalized RLU measured 4, 24, 48, and 72 hours h p.e. RBV and DMSOs were employed as positive and negative controls, respectively. The depicted values represent means+SD (E, F) or±SD (B-D) from 3 independent experiments. Abbreviations: h p.e., hours post-electroporation; RLU, relative light units; RBV, ribavirin; VC, vehicle control.

Cathepsin inhibitor K11777 impedes HEV infection

Cysteine proteases play pivotal roles in the entry process of several viruses, most notably Ebola²⁶ and SARS-CoV-2.²⁷ Interestingly, these cysteine proteases all belong to the CTS family, which are primarily localized in endo-lysosomal compartments and operate at acidic pH to digest incoming proteins. Blocking lysosomal acidification and thus CTS activity by ammonium chloride indeed strongly inhibited neHEV_CC infections (Figure 3A, B). To ascertain if CTSs are crucial host factors for HEV infection, we first confirmed the inhibitory potency of the broad-spectrum CTS inhibitor K11777 against recombinant CTSs by a cell-free enzyme assay. The inhibitor displayed an EC₅₀ of 0.9 µM, 3.3 µM, and 92 nM for CTSS, B and L, respectively, with CTSL showing the highest sensitivity (Figure 3C). In contrast, TLV required higher concentrations than K11777 to block CTS activity (Supplemental Figure S1, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). We next assessed the antiviral activity of K11777 in HEV infection experiments. In neHEV_CC-infected cells, K11777 exhibited an EC₅₀ of 764 pM and a CC₅₀ > 10 µM (Figure 3D, G), translating to a selectivity index of more than 10,000. Infection assays with the eHEV_CC particle showed a similar K11777 sensitivity compared to neHEV_CC, with an EC₅₀ of 18 pM for eHEV_CC (Figure 3E, G), implying a similar dependency of both HEV isoforms on CTSs. Furthermore, infection with the wild boar strain 83-2-27 demonstrated potent inhibition by K11777 with an EC₅₀ of 1.95 nM (Figure 3F, G), suggesting strain-independent antiviral activity of K11777. Also, an additive antiviral effect was observed in combination studies with RBV and K11777 (Supplemental Figure S2, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). Next, we investigated which viral life cycle step is perturbed by K11777. First, we determined replication capacity by HEV subgenomic reporter and found that similar to previously tested protease inhibitors, K11777 also did not affect virus replication (Supplemental Figure S3A, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). Next, to elucidate the involvement of CTSs in the viral entry process, we performed time-of-drug addition assays in which K11777 was introduced at various stages: preinfection (−1 hour), simultaneously during infection (0 hour), or postinfection (2, 4, 8, 12, 16, 24, 48, 72 hours) (Figure 3H). Notably, when introduced to cultures prior to infection as well as to cells already infected for up to 4 hours postinfection, K117777 retained its antiviral efficacy. However, a time-dependent loss in antiviral activity was observed when K11777 was administered for or beyond 8 hours postinfection, suggesting that K11777 targets the entry phase of the viral replication cycle and that CTSs are involved in virus entry. In contrast, a different pattern of viral inhibition was observed with the nucleoside analog RBV, a well-known inhibitor of HEV RNA replication. The suppression of HEV was reduced only when RBV was administered following the initiation of viral RNA replication observed when RBV was added after the onset of RNA virus replication (~24–72 hours p.i.), highlighting the distinct modes-of-action of K11777 and RBV in the HEV life cycle.

FIGURE 3.

Cathepsin inhibitor K11777 impedes HEV late entry with nanomolar efficacy. (A) Blocking lysosomal acidification by NH₄Cl inhibits HEV infection in hepatoma HepG2/C3A cells. Plotted are normalized (Norm.) infected cells in percent (%) and normalized cell viability in percent (%), determined for the treatment of 2.5 and 4 mM NH₄Cl. DMSO served as vehicle control (VC). To test the significance of mean differences, one-way ANOVA, followed by Dunnett multiple comparison test, was used. p-values >0.05 were considered to be not significant (ns). (B) Representative immunofluorescence images of Kernow-C1/p6 virus infected and NH₄Cl or DMSO (VC) treated cells. ORF2=green; DAPI=blue; scale bar=200 µm. (C) Recombinant CTSS, CTSB, and CTSL were incubated with up to 10 µM K11777 in a cell-free enzyme assay. (D–F) Dose-dependency of K11777 on nonenveloped cell culture-derived HEV (neHEV) infections (green data points) and cytotoxicity (gray data points) in hepatoma HepG2/C3A cells. Cells were infected with HEV in the presence of K11777 or vehicle control for 4 days before fixation, immunofluorescence staining, and analysis. Inhibition and cytotoxicity of Kernow-C1/p6 nonenveloped cell culture-derived HEV (D), eHEVcc (E), and wild boar 83-2-27 (F) strain by K11777. (G) Representative immunofluorescence images of 10 µM K11777 treatment in HEV infected cells. ORF2=green; DAPI=blue; scale bar=200 µm. (H, top) Experimental setup of the time-of-drug-addition assay. Hepatoma HepG2/C3A cells were inoculated with neHEV_CC at 0 hours and treated with DMSO, 0.1 µM K11777 or 25 µM RBV for 1 hour (−1) prior to, during (0) and 2, 4, 8, 12, 16, 24, 48, and 72 hours after infection until fixation at 96 hours after infection. At 8 hours after infection, inoculum was removed, and cells replenished with fresh medium containing drugs after several washes with PBS. (H, bottom) The inhibitory effect of K11777 on HEV_CC infection when added at different time points preinfection or postinfection is depicted by the light green curve. The broad-spectrum RNA virus inhibitor RBV served as positive control (gray curve). Data presented represents mean ± SD from 2 (C) or 3 independent experiments. Abbreviations: CTSB, cathepsin B; CTSL, cathepsin L; CTSS, cathepsin S; eHEV, enveloped form HEV; IF, immunofluorescence; MOI, multiplicity of infection; neHEV, nonenveloped cell culture-derived HEV; ORF, open reading frame; RBV, ribavirin; VC, vehicle control.

Cathepsin L is required for HEV infection

Next, we aimed to identify which specific CTS(s) are involved in HEV entry. Therefore, we first assessed the CTS expression patterns in liver tissue cells to provide a more comprehensive understanding of CTS expression in the liver. To this end, we analyzed single-cell RNA-sequencing data from the human liver cell atlas collected from nine healthy human donors (Supplemental Figure S4A, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). Notably, while most CTSs were expressed in the liver, CTSW, V, G, and E had reduced expression. Kupffer cells exhibited the highest average expression, followed by hepatocytes and stellate cells. Further examination of endogenous expression in primary human hepatocytes revealed prominent expression of CTSB, L, S, and Z (Figure 4A). A comparative analysis between HepG2 and Huh7.5 cells demonstrated only minor differences in reads per kilobase per million base pairs mapped levels and high levels of CTSB, C, and L in both cell lines (Figure 4B).

FIGURE 4.

Cathepsins are endogenously expressed in primary human liver cells and standard hepatoma cell lines. (A, B) Heat map of normalized transcript expression (reads per kilobase per million base pairs mapped [RPKM]) of CTSs in adult primary human hepatocytes (A) and hepatoma cell lines HepG2 (mean of n=4) and Huh7.5 (mean of n=6) (B). Abbreviations: CTSA, cathepsin A; CTSB, cathepsin B; CTSC, cathepsin C; CTSD, cathepsin D; CTSF, cathepsin F; CTSG, cathepsin G; CTSH, cathepsin H; CTSK, cathepsin K; CTSL, cathepsin L; CTSO, cathepsin O; CTSS, cathepsin S; CTSV, cathepsin V; CTSW, cathepsin W; CTSZ, cathepsin Z.

To investigate the role of specific cysteine CTSs in HEV infection, HepG2/C3A cells were treated with specific cysteine protease CTS inhibitors (Figure 5A–E). Notably, CTS inhibitors specifically targeting CTSB (CA-074) (Figure 5A), CTSC (Brensocatib) (Figure 5B), and CTSS (Petesicatib) (Figure 5C) did not have a discernible impact on either cell viability or HEV infectivity. In contrast, 2 inhibitors, CAA0225 (Figure 5D), a selective inhibitor of CTSL, and E64d (Figure 5E), an inhibitor of both CTSL and CTSB, significantly inhibited HEV infection, characterized by an EC₅₀ of 660 nM and 4 µM, respectively. To further confirm the importance of CTSL in HEV infection, we transfected HepG2/C3A cells with small interfering RNAs targeting CTSL or a nontargeting control (siCtrl) 48 hours prior to neHEV_CC inoculation. Notably, CTSL knockdown (Figure 5F) reduced neHEV_CC infection (Figure 5G, H) without affecting cell viability (Supplemental Figure S5, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). Furthermore, commercially obtained human HEK293T CTSL homozygous knockout cells were infected with neHEV_CC. Consistent with results obtained during CTSL knockdown, CTSL knockout (CTSL/KO) cells (Figure 5I) showed significant reduction in HEV infection, as evidenced by a reduced detection of the ORF2 capsid protein in immunofluorescence staining (Figure 5J, K). Conversely, CTSL overexpression in HEK293T wild type and HEK293T CTSL knockout cells (Figure 5I) led to a significant increase in neHEV_CC infections (Figure 5J, K). Notably, treatment with 1 µM K11777 in Kernow-C1/p6-Gaussia luciferase replicon transfected HEK293T-wild-type and HEK293T-knockout cells did not affect HEV replication (Supplemental Figure S3B, C, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). In sum, these results demonstrate that CTSL is required for HEV infection.

FIGURE 5.

CTSL inhibitors block HEV infection in vitro. (A–E) Inhibition of HEV infection by different doses of CA-074 (A), brensocatib (B), petesicatib (C), CAA0225 (D) and E64d (E) (green dots) and cell viability (gray dots) of cells treated with different doses of the drugs as indicated. Data depicts means±SD of 3 independent experiments. (F) Western blot analysis of lysates harvested from HepG2/C3A cells 48 hours after transfection with siCTSL or a siCtrl. (G) Representative immunofluorescence images of siCTSL or siCtrl transfected HepG2/C3A cells infected with nonenveloped cell culture-derived HEV 5 days post-transfection. ORF2=green; DAPI=blue; scale bar=200 µm. (H) Quantification of infected cells transfected with siCTSL normalized to cells transfected with siCtrl. Images from the center of each well were used for analysis in CellProfiler by setting a certain intensity threshold for detection of ORF2-positive cells. To test the significance of mean differences, two-tailed Wilcoxon test was used. p-values >0.05 were considered to be not significant (ns). (I) Expression of CTSL in HEK293T WT and CTSL-KO cells as well as CTSL overexpression in HEK293T WT/OX and CTSL-KO/OX cells determined by western blot. (J) Representative immunofluorescence images of HEK293T WT, HEK293T CTSL knockout and overexpression cells infected with nonenveloped cell culture-derived HEV. ORF2=green; DAPI=blue; scale bar=200 µm. (K) Effect of CTSL overexpression and knockout on HEV infection in HEK293T WT and CTSL KO cells, as determined by immunofluorescence staining against HEV ORF2 capsid protein. Data presented represents mean + SD from 3 independent experiments. To test the significance of mean differences, repeated measures one-way ANOVA, followed by Turkey multiple comparison test, were used. Abbreviations: CTSL, cathepsin L; KO, knockout; Norm., normalized; ORF, open reading frame; OX, overexpression; siCtrl, nontargeting control; siCTSL, siRNAs directed against CTSL; WT, wild type.

Cathepsin L mediates virus entry and cleaves glycosylated ORF2 protein as well as viral particles

Next, we confirmed the effect of cathepsins on neHEV_CC entry in S10-3 cells by an RNA fluorescence in situ hybridization entry assay.²⁸ We found a significant increase in capsid-associated genomes of neHEV_CC particles upon K11777 treatment compared to DMSO-treated S10-3 cells (Figure 6A–C). We also observed increased HEV capsid and genome co-localization 24 hours postinfection in cells treated with the specific CTSL inhibitor CAA0225, suggesting unsuccessful uncoating upon cathepsin L inhibition. To confirm the antiviral activity of both K11777 and CAA0225 in S10-3 cells, viral titer (neHEV_CC FFU) was quantified 5 days after infection (Supplemental Figure S6, Supplemental Digital Content 1, http://links.lww.com/HEP/I446). We observed that both inhibitors significantly reduced neHEV_CC infection when applied during the first hours of infection.

FIGURE 6.

CTSL is required for HEV cell entry and cleaves HEV ORF2 protein, generating unique cleavage fragments. (A) S10-3 cells were treated with either 0.1 µM K11777 or 10 µM CAA0225 and inoculated with nonenveloped cell culture-derived HEV. Twenty-four-hours after inoculation, cells were fixed, and HEV capsid and genomes were detected by immunofluorescence staining and RNA fluorescence in situ hybridization using an ORF1 probe, respectively. Fluorescence images are representative of n=3. ORF2=green; DAPI=blue. (B) Line graphs show the fluorescence intensities of HEV genomes and green fluorescence protein measured across the region of interest indicated by the white line in A. (C) Depicted are the calculated percentages of RNA particles associated with capsids out of the total number of detected genomes per cell. HEV genomes and capsids were quantified using CellProfiler. Statistical analysis was performed by repeated measures one-way ANOVA, followed by Dunnett multiple comparison test. p-values >0.05 were considered to be not significant (ns). (D) Analysis of CTSL-mediated ORF2-protein cleavage. Glycosylated pORF2 (4 µg) was incubated in the presence or absence of 10 µg/mL CTSL for 14 hours at 25°C. The reaction was further supplemented with 1 µM K11777 or DMSO, as indicated. Proteins were subjected to SDS-PAGE and detected by Coomassie blue staining. (E) Representative fluorescence images of HepG2/C3A cells either inoculated with Purified HEV particles (5µL of 4.36 × 10⁵ FFU/mL stock) or PBS. ORF2=green; DAPI=blue. (F) Cleavage of purified infectious HEV particle by CTSL. 4.36×10⁵ FFU/mL of purified HEV particles was incubated in the presence or absence of 10 µg/mL CTSL for 14 hours at 25°C. The reaction was further supplemented with 1 µM K11777 or DMSO, as indicated. Representative blots in D and F are from at least 2 independent experiments. Abbreviations: CTSL, cathepsin L; ORF, open reading frame; VC, vehicle control.

To explore if CTSL cleaves the ORF2 protein, we conducted in vitro cleavage assays using the glycosylated ORF2 protein (~61 kDa) and recombinant CTSL protein (~35 kDa) (Figure 6D). Incubating glycosylated ORF2 with CTSL led to the effective cleavage of the ORF2 protein into smaller products, specifically fragments around 58–61 kDa (3 fragments), around 25 kDa (2 fragments), and a single fragment of approximately 18 kDa. Cleavage experiments with gradient-purified infectious viral particles (Figure 6E) resulted in two smaller cleavage products (~70 and ~ 60 kDa) (Figure 6F). Notably, the cleavage activity of CTSL was blocked by applying 1 µM of K11777 to both the ORF2 protein and viral particle reaction mix (Figure 6D, F). These findings suggest that CTSL efficiently cleaves the ORF2 protein and HEV particles into smaller protein fragments.

Cathepsins are critical for HEV infection in HepaRG cells and primary human hepatocytes

To characterize the role of CTSs in a more authentic cell culture model, we assessed their significance for HEV infection in differentiated HepaRG cells (Figure 7A, B). These hepatic cells emulate many features of mature hepatocytes, such as high plasticity and bile canaliculi formation. We confirmed that K11777 inhibited HEV infections in HepaRGs (Figure 7A) with an EC₅₀ of 5.8 nM (Figure 7B). To further address the importance of CTSs in primary liver cultures, we infected PHH with neHEV_CC in the presence of K11777. Our data revealed a dose-dependent HEV inhibition by K11777 (Figure 7C, D) with almost no detectable ORF2-positive cells when treated with 10 µM K11777 (Figure 7E). Taken together, these data indicate that CTSs are critical for HEV infection in primary cells and that HEV infection can be effectively restricted by the application of the K11777 inhibitor during inoculation.

FIGURE 7.

K11777 inhibits HEV infection in HepaRG cells and primary human hepatocytes. (A, B) Differentiated HepaRG cells were infected with nonenveloped cell culture-derived HEV and incubated in the presence of K11777 or vehicle control (VC) for 4 days until immunofluorescence staining against ORF2 capsid protein. (A) Representative immunofluorescence images display the effect of 1 µM K11777 on the infectivity of nonenveloped cell culture-derived HEV. ORF2=green; DAPI=blue. (B) Dose-response curves of K11777 on nonenveloped cell culture-derived HEV infections (green data points) and cell viability (gray data points), and normalized to VC, in HepaRG cells. Shown are means±SD of 3 independent experiments. (C) Primary human hepatocytes were infected with Kernow-C1/p6 virus and treated with 0.1, 1, and 10 µM K11777 for 3 days. Treatment with DMSO and 25 µM RBV served as negative and positive control, respectively. (D) Cell viability was determined by LDH release assay. Data depicts means+SD of 3 independent experiments. To test the significance of mean differences, one-way ANOVA, followed by Dunnett multiple comparison test, was used. p-values >0.05 were considered to be not significant (ns). (E) Representative immunofluorescence images. ORF2=green; DAPI=blue; scale bar=200 µm. Abbreviations: CTSL, cathepsin L; ORF, open reading frame; RBV, ribavirin; VC, vehicle control.

DISCUSSION

Although cell entry mechanisms used by viruses are highly diverse, many general principles of virus entry are conserved: viruses attach to target cells, internalize, cross a lipid bilayer, and deliver their nucleic acid to an intracellular site for replication. For HEV, these entry mechanisms remain poorly understood, largely due to the absence of a robust in vitro cell culture system to study HEV infections in the past decades. In this study, we used an advanced cell culture system to demonstrate the pivotal role of cysteine proteases, especially CTSs, in the entry of both neHEV_CC and eHEV_CC. Moreover, we provided experimental evidence highlighting CTSL as an essential host factor for HEV entry and demonstrate that the pan-cathepsin inhibitor K11777 significantly reduces HEV infection in both hepatoma and primary cells.

First, we observed that the HCV NS3/4A replication inhibitor TLV is effective against HEV infection, but not HEV RNA replication. This absence of observed reduction in HEV RNA replication upon TLV treatment led us to hypothesize that TLV does not target a putative HEV protease but rather a host cellular protease. Indeed, current evidence indicates that in addition to direct viral targets such as HCV NS3/4A and SARS-CoV-2 M^pro,^29,30 TLV might also affect indirect host targets, including CTSL.³⁰ Notably, TLV did not potently inhibit CTSL in our in vitro enzyme assay, which could potentially be explained by the necessity for higher concentrations of TLV. Additionally, TLV might inhibit HEV infection not through the inhibition of CTSL but rather by targeting other cathepsins that are necessary to establish infection. Another indication of CTS targeting by TLV stems from its structure. TLV is a linear α-ketoamide inhibitor that covalently binds to the catalytic serine (S139) of NS3/4A through its α-ketoamide warhead.³¹ These α-ketoamide motifs are frequently incorporated in lead molecules targeting serine and cysteine proteases.³² In fact, small molecules bearing an α-ketoamide warhead have been shown to inhibit CTSS potently at low nanomolar concentrations.³³ In a similar vein, TLV’s α-ketoamide motif could potentially bind to CTSs, inhibiting their activity; however, this warrants further investigation.

Next, we evaluated inhibitors targeting 3 primary classes of cellular proteases: cysteine, serine, and aspartyl proteases. Notably, we observed significant inhibition of HEV infection with leupeptin (EC₅₀=1.7 µM), a combined serine and cysteine protease inhibitor. However, both serine and aspartyl protease inhibitors did not reduce HEV infections. Similar to observations with TLV, HEV RNA replication was not impaired upon leupeptin treatment, further highlighting the role of cellular proteases in HEV infection rather than targeting any putative protease domains encoded by the virus genome. Additionally, leupeptin has also been described as a potent “tight binding” inhibitor of CTSL,³⁴ which aligns with our findings that highlight the significance of CTSL in HEV infection.

Our data also suggests that neHEV_CC infection depends on lysosomal acidification, a condition required for CTS activity. Treatment with NH₄Cl, an agent that neutralizes endosomal pH, reduced HEV infectivity, which is in line with a recent study by Fu et al,²⁸ who found that bafilomycin A, another endosomal acidification inhibitor was able to block neHEV_CC infection. However, these results contradict the findings of a study by Yin et al,¹⁶ which indicated that neHEV_CC does not require acidification of the endosome. The reason for this discrepancy may be attributed to different experimental setups, necessitating further independent experimental investigations for clarification.

Based on the aforementioned results and the fact that CTSs are highly expressed and the main lysosomal proteases involved in protein degradation,³⁵ we investigated the role of different cysteine CTSs (CTSB, CTSC, CTSS, and CTSL). We found that the CTSL-specific inhibitor, CAA0225, reduced HEV infectivity with an EC₅₀ of 660 nM, indicating a role of CTSL in HEV infection. Fittingly, we noted high CTSL expression in human hepatoma cells, primary human hepatocytes, and liver tissue, further suggesting its potential role in HEV entry. To date, several viruses have been shown to rely on CTSs for effective cell entry, such as Ebola virus, SARS-CoV-2, and reovirus. For instance, CTSL and B cleave the Ebola virus glycoprotein GP1, facilitating its interaction with cellular receptor(s) and viral entry,^36,37 while the SARS-CoV-2 spike protein is cleaved by CTSB and L³⁸ initiating membrane fusion and uncoating of the viral RNA. CTSs, crucial for reovirus entry, proteolytically process the capsid protein leading to structural rearrangements that eventually result in the release of transcriptionally active viral core into the cytoplasm (reviewed in the study by Mainou and Dermody³⁹) Reovirus can employ various CTSs (CTSB, CTSL, and CTSS) for its processing,^40,41 which is believed to contribute to its wide host tropism. By analogy, HEV might also use multiple different CTSs for its ORF2 capsid processing, explaining why HEV infection was not completely abrogated in both CTSL knockdown and knockout cells. Concurrently, this phenotype in CTSL knockdown and knockout cells might be explained by the potential use of different entry strategies by HEV (similar to that observed for SARS-CoV-2). Also, HEV tropism is very broad, as evidenced by the fact that HEV is able to infect neuronal,⁴² placental,⁴³ and human intestinal cells.⁴⁴ Therefore, it will be of interest to determine if this broad cell tropism is, in part, a product of the ability to engage different CTSs. Furthermore, considering that our experiments employed HEK293T cells for the CTSL gene knockout, it remains to be determined whether the knockout of the CTSL gene results in reduced HEV infection in liver cells.

Next, we explored the stage of the viral life cycle targeted by K11777 through conducting time-of-addition and RNAscope analysis. Both methods confirmed that HEV entry and the dissociation of viral RNA from the capsid are inhibited by this compound, highlighting the necessity of cathepsins for viral entry. Additionally, we carried out in vitro cleavage assays with the glycosylated ORF2 protein and viral particles and found that CTSL was capable of cleaving both the ORF2 protein and virus particles into smaller fragments. This indicates potential cleavage sites within the ORF2 protein and suggests that CTSL cleaves the capsid. A recent study by Nishiyama and colleagues revealed that the C-terminal region of the virion-associated HEV ORF2 protein found in human feces is truncated by intestinal proteases. This truncation results in a reduced molecular weight of the capsid, similar to that observed in detergent-treated, protease-digested HEV_CC virions.⁴⁵ For several viruses, including poliovirus, rotavirus, and norovirus, capsid proteins are known to undergo proteolytic modifications by gastrointestinal tract proteases. These modifications play a crucial role in capsid maturation, stability, and infectivity. Similarly, it is plausible that truncation of the ORF2 C-terminal region, particularly the P-domain—which forms a dimeric spike essential for attachment to susceptible cells—may prepare the capsid protein for successful host cell binding. This suggests a mechanism that primes the virus for entry rather than circumventing the need for cathepsins through proteolytic processing of the HEV capsid by gastrointestinal proteases during neHEV entry. However, further studies are required to elucidate how this potential extracellular processing in the gastrointestinal tract and the suggested intracellular cleavage by CTS are related to the HEV life cycle.

Given that the data obtained in this study suggests that CTS activity plays a critical role in HEV entry into liver cells, targeting CTSs may represent an attractive targeting strategy for the development of new HEV antiviral drugs. The pan-specific CTS inhibitor, K11777, markedly reduced infectivity of both neHEV_CC and eHEV_CC as well as the wild boar strain 83-2-27 at pico- to nanomolar concentrations, confirming the importance of CTSs for both HEV isoforms and strains. The EC₅₀ values for eHEV_CC were even 10-fold lower compared to neHEV_CC in HepG2/C3A cells. Moreover, we have shown that K11777 inhibited a broad panel of CTSs and that this translated to a high potency in cell culture and notably in primary human hepatocytes. A higher concentration of K11777 was required to inhibit HEV in PHH compared to nonprimary cell lines. This disparity could stem from a reduced uptake and metabolism of the compound in PHH or the possibility that other cathepsins (CTS), not inhibited by K11777, may facilitate HEV entry in PHH. As we determined an additive antiviral effect when combined with RBV, whether combination therapy is able to increase efficacy and reduce reduce the occurrence of resistance remains to be explored in future in vivo assays. K11777 has originally been developed against Trypanosoma cruzei ⁴⁶ but has shown effective against several Filo- and coronaviruses,^47,48 including SARS-CoV-2.⁴⁹ Despite not being FDA-approved, it is orally available and currently undergoing a phase-II clinical trial against SARS-CoV-2 under the name SLV213,⁴⁹ which is promising with respect to safety and tolerability. Future in vivo studies are essential to validate these encouraging findings and potentially expand the arsenal of effective HEV antivirals.

In conclusion, while HEV entry remains poorly understood, a process wherein HEV ORF2 undergoes CTSL-mediated proteolytic processing, resulting in viral RNA cytosolic release, appears plausible. However, further studies will be essential to define putative interactions between the HEV virion and CTSs. Finally, the CTS inhibitor K11777 emerged as a potent compound providing both excellent efficacy and selectivity in vitro.