Inhibit Hepatitis C Virus Infection Research Articles

Virtual Screening of Telaprevir and Danoprevir Derivatives for Hepatitis C Virus NS3/4A Protease Inhibitors

Background: The NS3/4A protease is a common target for inhibiting hepatitis C virus (HCV) infection. Telaprevir and danoprevir have promising activity in combating these virus-associated infections and are used as HCV protease inhibitors. Objective: In this study, we have found different tested derivative compounds for developing various HCV NS3/4A protease inhibitors by designing the chemical structures of telaprevir and danoprevir. Methods: In silico studies were carried out to find better drug candidatures from these derivative compounds. The docking studies were performed on HCV NS3/4A protease receptors (PDB: 3SV6 & 5EQR) using Autodock vina. DFT, global reactivity, ADME (Absorption, distribution, metabolism & excretion), and toxicity analysis were also performed for these designed compounds. The stability of the proteinligand complexes was quantified by MD simulation and MMPBSA studies. Results: 16 derivatives (four as telaprevir and twelve as danoprevir) have satisfied higher binding affinity of interaction with NS3/4A protease, compared to telaprevir and danoprevir. These compounds have also passed all rules of drug candidature to serve as the best HCV inhibitors. Conclusion: These 16 ligands can be effective inhibitors against HCV NS3/4A protease. These ligands must obey the drug candidate behavior by in vitro and in vivo analysis to inhibit HCV infection.

Hepatitis C virus non-structural proteins modulate cellular kinases for increased cytoplasmic abundance of host factor HuR and facilitate viral replication.

Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3'UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-δ inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-δ and activators of AMPK, either separately or in combination, to inhibit HCV infection.

Dual roles and potential applications of exosomes in HCV infections.

The hepatitis C virus (HCV) causes severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma, which have high morbidity and mortality. Antibody targeting receptor-mediated HCV infections have limited therapeutic benefits, suggesting that the transmission of HCV infections is possibly mediated via receptor-independent mechanisms. Exosomes are membrane-enclosed vesicles with a diameter of 30-200 nm, which originate from the fusion of endosomal multivesicular bodies with the plasma membrane. Accumulating evidence suggests that exosomes have a pivotal role in HCV infections. Exosomes can transfer viral and cellular bioactive substances, including nucleic acids and proteins, to uninfected cells, thus spreading the infection by masking these materials from immunological recognition. In addition, exosomes originating from some cells can deliver antiviral molecules or prompt the immune response to inhibit HCV infection. Exosomes can be used for the diagnosis of HCV-related diseases, and are being presently evaluated as therapeutic tools for anti-HCV drug delivery. This review summarizes the current knowledge on the dual roles and potential clinical applications of exosomes in HCV infections.

Natural flavonoids effectively block the CD81 receptor of hepatocytes and inhibit HCV infection: a computational drug development approach.

Hepatitis C virus (HCV) infection is a major public health concern, and almost two million people are infected per year globally. This is occurred by the diverse spectrum of viral genotypes, which are directly associated with chronic liver disease (fibrosis, and cirrhosis). Indeed, the viral genome encodes three principal proteins as sequentially core, E1, and E2. Both E1 and E2 proteins play a crucial role in the attachment of the host system, but E2 plays a more fundamental role in attachment. The researchers have found the "E2-CD81 complex" at the entry site, and therefore, CD81 is the key receptor for HCV entrance in both humans, and chimpanzees. So, the researchers are trying to block the host CD81 receptor and halt the virus entry within the cellular system via plant-derived compounds. Perhaps that is why the current research protocol is designed to perform an in silico analysis of the flavonoid compounds for targeting the tetraspanin CD81 receptor of hepatocytes. To find out the best flavonoid compounds from our library, web-based tools (Swiss ADME, pKCSM), as well as computerized tools like the PyRx, PyMOL, BIOVIA Discovery Studio Visualizer, Ligplot+ V2.2, and YASARA were employed. For molecular docking studies, the flavonoid compounds docked with the targeted CD81 protein, and herein, the best-outperformed compounds are Taxifolin, Myricetin, Puerarin, Quercetin, and (-)-Epicatechin, and outstanding binding affinities are sequentially -7.5, -7.9, -8.2, -8.4, and -8.5kcal/mol, respectively. These compounds have possessed more interactions with the targeted protein. To validate the post docking data, we analyzed both 100ns molecular dynamic simulation, and MM-PBSA via the YASARA simulator, and finally finds the more significant outcomes. It is concluded that in the future, these compounds may become one of the most important alternative antiviral agents in the fight against HCV infection. It is suggested that further in vivo, and in vitro research studies should be done to support the conclusions of this in silico research workflow.

MxB Disrupts Hepatitis C Virus NS5A-CypA Complex: Insights From a Combined Theoretical and Experimental Approach.

The human myxovirus resistance B (MxB) protein is an interferon-induced restriction factor that fights a wide range of viruses. We previously demonstrated that MxB binds to hepatitis C virus (HCV)-encoded non-structural protein 5A (NS5A) and inhibits HCV infection by impairing the formation of cyclophilin A (CypA)–NS5A complex. However, the molecular details about how the presence of MxB diminishes the binding of NS5A to CypA remain uncovered. In this study, through molecular dynamic simulations and biochemical assays, we characterized that MxB binds to NS5A domain I through its N-terminal and GTPase domains. Specifically, amino acids (aa.) 189–191 and aa. 330–334 within MxB, together with NS5A residues aa. 71–73, are crucial for MxB–NS5A interaction. Furthermore, we predicted the CypA:NS5A and CypA:NS5A:MxB complexes and calculated the per-residue energy decomposition for identified key residues of the CypA–NS5A interface. A 28% decrease in CypA–NS5A binding affinity was observed in the presence of MxB, suggesting a weakened CypA–NS5A association upon binding of MxB to NS5A, which may contribute to the MxB-mediated inhibitory effect on the formation of CypA–NS5A complex. This work provides information for the antiviral mechanism of MxB and may facilitate the discovery of new strategies to combat CypA-dependent viruses.

Role of Ras-related Nuclear Protein/Polypyrimidine Tract Binding Protein in Facilitating the Replication of Hepatitis C Virus.

Background and AimsRas-related nuclear (RAN) protein is a small GTP-binding protein that is indispensable for the translocation of RNA and proteins through the nuclear pore complex. Recent studies have indicated that RAN plays an important role in virus infection. However, the role of RAN in hepatitis C virus (HCV) infection is unclear. The objective of this study was to investigate the role and underlying mechanisms of RAN in HCV infection.MethodsHuh7.5.1 cells were infected with the JC1-Luc virus for 24 h and then were incubated with complete medium for an additional 48 h. HCV infection and RAN expression were determined using luciferase assay, quantitative reverse transcription-PCR and western blotting. Small interfering RNA was used to silence RAN. Western blotting and immunofluorescence were used to evaluate the cytoplasmic translocation of polypyrimidine tract-binding (PTB), and coimmunoprecipitation was used to examine the interaction between RAN and PTB.ResultsHCV infection significantly induced RAN expression and cytoplasmic redistribution of PTB. Knockdown of RAN dramatically inhibited HCV infection and the cytoplasmic accumulation of PTB. Colocalization of RAN and PTB was determined by immunofluorescence, and a direct interaction of RAN with PTB was demonstrated by coimmunoprecipitation.ConclusionsPTB in the host cytoplasm is directly associated with HCV replication. These findings demonstrate that the involvement of RAN in HCV infection is mediated by influencing the cytoplasmic translocation of PTB.

Inhibitory activity of limonoids from Khaya grandifoliola C.DC (Meliaceae) against hepatitis C virus infection in vitro.

Objective:A fraction from Khaya grandifoliola has recently been shown to inhibit hepatitis C virus (HCV) infection and three limonoids (17-epi-methyl-6-hydroxylangolensate, 7-deacetoxy-7-oxogedunin and 7-deacetoxy-7R-hydroxygedunin) were purified from this fraction. The present study aimed at assessing the inhibitory effect of these limonoids on HCV using cell-culture derived HCV (HCVcc) system. Materials and Methods:Cytotoxic effects of the limonoids on Huh7.5 cells were assessed by MTT assay. Huh7.5 cells were transfected with RNA transcripts of the plasmid Jc1/GLuc2a, carrying a Gaussia luciferase reporter gene to rescue the HCVcc particles which were used to infect naïve cells in the presence or absence of the studied limonoids during 72 hr. Infection and replication rates were monitored by luciferase reporter assay and immunofluorescence assay (IFA) while cellular gene expression was analyzed by western blot, respectively.Results:The limonoids inhibited HCV infection mostly by targeting entry and replication stage. Their inhibitory effect on entry step, comparable to that of anti-CD81 antibody, was related to the blocking of CD81 receptor. In the replication step, the limonoids decreased the expression of NS5B similar to danoprevir. These compounds also significantly decreased but up-regulated the expression of Class-III phosphatidylinositol 4-kinase alpha and 2’,5’-oligoadenylate synthase-3, respectively. Conclusion:The present findings suggest that limonoids from K. grandifoliola are potential anti-HCV agents and may offer an advantage in the treatment of HCV infection.

Potent virucidal activity of honeybee “Apis mellifera” venom against Hepatitis C Virus

Hepatitis C virus (HCV) is a global viral widespread without an available vaccine to prevent infection. HCV infection can cause serious liver diseases such as hepatocellular carcinoma (HCC). Current treatment of HCV infection depends on the FDA approved direct-acting antivirals (DAAs) which have side effects and expensive. Thus, development of a novel, more efficient, along with affordable pricing anti-HCV agents is still required. The purpose of the present study is to evaluate the antiviral effects of bee venom (BV) from the honeybee Apis mellifera on the HCV replication life cycle. The crude venom and its components were examined for their anti-HCV activities using Huh7it-1 cultured cells and the JFH1 strain of HCV genotype 2a. Results revealed that BV inhibited HCV infection with 50% inhibitory concentration (IC50) of 0.05 ng/ml, while the 50% cytotoxic concentration (CC50) being 20,000 ng/ml. The venom directly blocked HCV/cell entry by acting on virus particles in a dose dependent manner, whereas no interference on the host cells. Furthermore, venom showed no inhibitory effect on HCV replication and release. Interestingly, none of the main BV components including the mast cell degranulating peptide (MCD), mpamin, or the small peptides melittin (MLT) showed anti-HCV activity up to 5 μg/ml. In conclusion, these results suggest that BV has a direct virucidal activity against HCV and may exert its antiviral effect through a non-common peptide(s) or toxin complex within the crude venom. Therefore, the crude BV can be considered as a promising candidate for characterization and development of new and natural anti-HCV therapeutic agents.

The Mechanism of Suppression of Chronic Hepatitis C Infection by the Medicine Stimforte

The effect of the drug Stimforte on infection by the Hepatitis C virus (HCV) has been studied. Stimforte partially inhibits HCV infection at a dose of 100 μg/mouse and almost completely at a dose of 300 μg/mouse within 24 h after administration of the drug. The mice sera resulting after 24 h in the presence of 100 and 300 µg/mouse of Stimforte effectively inhibit the production of HCV. Doses of 150, 200, and 250 µg/mouse are not effective. Stimulation of interferon-β (IFN-β) production is only observed at doses of 100 and 300 µg/mouse, which explains well the neutralizing capacity of the sera. The amount of IFN-γ also correlates well with the antiviral activity and neutralizing activity of mice sera. The drug practically does not stimulate production of IFN-λ. Thus, the neutralizing activity of sera and the antiviral activity are largely determined by the 1st and 2nd IFN groups.

Inhibitory Activity of a Scorpion Defensin BmKDfsin3 against Hepatitis C Virus.

Hepatitis C virus (HCV) infection is a major worldwide health problem which can cause chronic hepatitis, liver fibrosis and hepatocellular carcinoma (HCC). There is still no vaccine to prevent HCV infection. Currently, the clinical treatment of HCV infection mainly relies on the use of direct-acting antivirals (DAAs) which are expensive and have side effects. Here, BmKDfsin3, a scorpion defensin from the venom of Mesobuthus martensii Karsch, is found to dose-dependently inhibit HCV infection at noncytotoxic concentrations and affect viral attachment and post-entry in HCV life cycle. Further experimental results show that BmKDfsin3 not only suppresses p38 mitogen-activated protein kinase (MAPK) activation of HCV-infected Huh7.5.1 cells, but also inhibits p38 activation of Huh7.5.1 cells stimulated by tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or lipopolysaccharide (LPS). BmKDfsin3 is also revealed to enter into cells. Using an upstream MyD88 dimerization inhibitor ST2345 or kinase IRAK-1/4 inhibitor I, the inhibition of p38 activation represses HCV replication in vitro. Taken together, a scorpion defensin BmKDfsin3 inhibits HCV replication, related to regulated p38 MAPK activation.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus.

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.

Inhibition Mechanisms of Hepatitis C Virus Infection by Caffeic Acid and Tannic Acid.

Previously, we reported that coffee extract and its constituents, caffeic acid (CA) and p-coumaric acid, inhibit infection by the hepatitis C virus (HCV). In the present report, we identified another coffee-related compound, tannic acid (TA), which also inhibits HCV infection. We systematically evaluated which steps of the viral lifecycle were affected by CA and TA. TA substantially inhibits HCV RNA replication and egression, while CA does not. The infectivity of the HCV pretreated with CA or TA was almost lost. Cellular attachment of HCV particles and their interaction with apolipoprotein E, which is essential for HCV infectivity, were significantly reduced by CA. These results indicate that CA inhibits HCV entry via its direct effect on viral particles and TA inhibits HCV RNA replication and particle egression as well as entry into host cells. Taken together, our findings may provide insights into CA and TA as potential anti-HCV strategies.

A central hydrophobic E1 region controls the pH range of hepatitis C virus membrane fusion and susceptibility to fusion inhibitors

Hepatitis C virus (HCV) infection causes chronic liver disease. Antivirals have been developed and cure infection. However, resistance can emerge and salvage therapies with alternative modes of action could be useful. Several licensed drugs have emerged as HCV entry inhibitors and are thus candidates for drug repurposing. We aimed to dissect their mode of action, identify improved derivatives and determine their viral targets. HCV entry inhibition was tested for a panel of structurally related compounds, using chimeric viruses representing diverse genotypes, in addition to viruses containing previously determined resistance mutations. Chemical modeling and synthesis identified improved derivatives, while generation of susceptible and non-susceptible chimeric viruses pinpointed E1 determinants of compound sensitivity. Molecules of the diphenylpiperazine, diphenylpiperidine, phenothiazine, thioxanthene, and cycloheptenepiperidine chemotypes inhibit HCV infection by interfering with membrane fusion. These molecules and a novel p-methoxy-flunarizine derivative with improved efficacy preferentially inhibit genotype 2 viral strains. Viral residues within a central hydrophobic region of E1 (residues 290-312) control susceptibility. At the same time, viral features in this region also govern pH-dependence of viral membrane fusion. Small molecules from different chemotypes related to flunarizine preferentially inhibit HCV genotype 2 membrane fusion. A hydrophobic region proximal to the putative fusion loop controls sensitivity to these drugs and the pH range of membrane fusion. An algorithm considering viral features in this region predicts viral sensitivity to membrane fusion inhibitors. Resistance to flunarizine correlates with more relaxed pH requirements for fusion. This study describes diverse compounds that act as HCV membrane fusion inhibitors. It defines viral properties that determine sensitivity to these molecules and thus provides information to identify patients that may benefit from treatment with membrane fusion inhibitors.

A Long Noncoding RNA Regulates Hepatitis C Virus Infection Through Interferon Alpha-Inducible Protein 6.

Long noncoding RNAs (lncRNAs) play a critical role in the regulation of many important cellular processes. However, the mechanisms by which lncRNAs regulate viral infection and host immune responses are not well understood. We sought to explore lncRNA regulation of hepatitis C virus (HCV) infection and interferon response. We performed RNA sequencing (RNAseq) in Huh7.5.1 cells with or without interferon alpha (IFNα) treatment. Clustered regularly interspaced short palindromic repeats/Cas9 guide RNA (gRNA) was used to knock out selected genes. The promoter clones were constructed, and the activity of related interferon-stimulated genes (ISGs) were detected by the secrete-pair dual luminescence assay. We constructed the full-length and four deletion mutants of an interferon-induced lncRNA RP11-288L9.4 (lncRNA-IFI6) based on predicted secondary structure. Selected gene mRNAs and their proteins, together with HCV infection, in Huh7.5.1 cells and primary human hepatocytes (PHHs) were monitored by quantitative real-time PCR (qRT-PCR) and western blot. We obtained 7,901 lncRNAs from RNAseq. A total of 1,062 host-encoded lncRNAs were significantly differentially regulated by IFNα treatment. We found that lncRNA-IFI6 gRNA significantly inhibited HCV infection compared with negative gRNA control. The expression of the antiviral ISG IFI6 was significantly increased following lncRNA-IFI6 gRNA editing compared with negative gRNA control in Japanese fulminant hepatitis 1 (JFH1)-infected Huh7.5.1 cells and PHHs. We observed that lncRNA-IFI6 regulation of HCV was independent of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. lncRNA-IFI6 negatively regulated IFI6 promoter function through histone modification. Overexpression of the truncated spatial domain or full-length lncRNA-IFI6 inhibited IFI6 expression and increased HCV replication. Conclusion: A lncRNA, lncRNA-IFI6, regulates antiviral innate immunity in the JFH1 HCV infection model. lncRNA-IFI6 regulates HCV infection independently of the JAK-STAT pathway. lncRNA-IFI6 exerts its regulatory function via promoter activation and histone modification of IFI6 through its spatial domain.

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus in cell culture.

The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3’-monogallate (TF2), and theaflavin-3-3’-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.

Methanolic Extract of Rhizoma Coptidis Inhibits the Early Viral Entry Steps of Hepatitis C Virus Infection.

Hepatitis C Virus (HCV) remains an important public health threat with approximately 170 million carriers worldwide who are at risk of developing hepatitis C-associated end-stage liver diseases. Despite improvement of HCV treatment using the novel direct-acting antivirals (DAAs) targeting viral replication, there is a lack of prophylactic measures for protection against HCV infection. Identifying novel antivirals such as those that target viral entry could help broaden the therapeutic arsenal against HCV. Herein, we investigated the anti-HCV activity of the methanolic extract from Rhizoma coptidis (RC), a widely used traditional Chinese medicine documented by the WHO and experimentally reported to possess several pharmacological functions including antiviral effects. Using the cell culture-derived HCV system, we demonstrated that RC dose-dependently inhibited HCV infection of Huh-7.5 cells at non-cytotoxic concentrations. In particular, RC blocked HCV attachment and entry/fusion into the host cells without exerting any significant effect on the cell-free viral particles or modulating key host cell entry factors to HCV. Moreover, RC robustly suppressed HCV pseudoparticles infection of Huh-7.5 cells and impeded infection by several HCV genotypes. Collectively, our results identified RC as a potent antagonist to HCV entry with potential pan-genotypic properties, which deserves further evaluation for use as an anti-HCV agent.

A novel occludin-targeting monoclonal antibody prevents hepatitis C virus infection in vitro

Since hepatitis C virus (HCV) is thought to enter into host hepatocytes using the same cellular pathways regardless of the genotypes, the host factors are promising targets to prevent and treat HCV infection. Human occludin (hOCLN) is one representative entry factor, and its second extracellular loop (EC2) contributes to the species selectivity of HCV-susceptibility. However, the exact function of hOCLN during HCV entry remains unknown, and no hOCLN-targeting antibodies or synthetic drugs that prevent and treat HCV infection have yet been developed. Here we generated the anti-hOCLN-EC2 monoclonal antibody (mAb) 67-2, and demonstrated that it efficiently inhibited HCV infection in the HCV-permissive human cell line Huh7.5.1. We also showed, using three different culture systems of Huh7.5.1 cells, that this novel mAb is accessible to OCLN from the basolateral side of hepatocytes but not from the apical side. In addition, our Western blot analyses indicated that the established 67-2 mAb reacted not only with hOCLN but also with mouse OCLN, strongly suggesting that 67-2 does not recognize the human-specific amino acids in OCLN-EC2. Moreover, we revealed that the anti-hOCLN-EC2 mAb 67-2 showed no adverse effects on cell viability or the barrier function of tight junctions.

Infection with hepatitis C virus depends on TACSTD2, a regulator of claudin-1 and occludin highly downregulated in hepatocellular carcinoma.

Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.

A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism.

Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.