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Drug and target discovery for advanced liver disease are hampered by a lack of suitable models for clinical translation. Here the authors present a human liver cell-based system modeling a clinical prognostic signature allowing to propose nizatidine for treatment of advanced liver fibrosis and hepatocellular carcinoma prevention.

Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder due to increased accumulation of fat in the liver and in many cases to enhanced inflammation. Although the contribution of inflammation in the pathogenesis of NAFLD is well established, the cytokines that are involved and how they influence liver transformation are still poorly characterized. In addition, with other modifiers, inflammation influences NAFLD progression to liver cirrhosis and hepatocellular carcinoma, demonstrating the need to find new molecular targets with potential future therapeutic applications. We investigated gene signatures in 38 liver biopsies from patients with NAFLD and obesity who had received bariatric surgery and compared these to 10 control patients who had received a cholecystectomy, using DNA microarray technology. A subset of differentially expressed genes was then validated on a larger cohort of 103 patients who had received bariatric surgery for obesity; data were thoroughly analyzed in terms of correlations with NAFLD pathophysiological parameters. Finally, the impact of a specific cytokine, interleukin‐32 (IL32), was addressed on primary human hepatocytes (PHHs). Transcript analysis revealed an up‐regulation of proinflammatory cytokines IL32, chemokine (C‐X‐C motif) ligand 9 (CXCL9), and CXCL10 and of ubiquitin D (UBD), whereas down‐regulation of insulin‐like growth factor‐binding protein 2 (IGFBP2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) was reported in patients with NAFLD. Moreover, IL32, which is the major deregulated gene, correlated with body mass index (BMI), waist circumference, NAFLD activity score (NAS), aminotransferases (alanine aminotransferase [ALAT] and aspartate aminotransferase [ASAT]), and homeostasis model assessment of insulin resistance (HOMA‐IR) index in patients. Consistent with an instrumental role in the pathophysiology of NAFLD, treatment of control human hepatocytes with recombinant IL32 leads to insulin resistance, a hallmark metabolic deregulation in NAFLD hepatocytes. Conclusion: IL32 has a critical role in the pathogenesis of NAFLD and could be considered as a therapeutic target in patients.

Summary: Chronic hepatitis B, C, and D virus (HBV, HCV, and HDV) infections are the leading causes of liver disease and cancer worldwide. Recently, the solute carrier and sodium taurocholate co-transporter NTCP has been identified as a receptor for HBV and HDV. Here, we uncover NTCP as a host factor regulating HCV infection. Using gain- and loss-of-function studies, we show that NTCP mediates HCV infection of hepatocytes and is relevant for cell-to-cell transmission. NTCP regulates HCV infection by augmenting the bile-acid-mediated repression of interferon-stimulated genes (ISGs), including IFITM3. In conclusion, our results uncover NTCP as a mediator of innate antiviral immune responses in the liver, and they establish a role for NTCP in the infection process of multiple viruses via distinct mechanisms. Collectively, our findings suggest a role for solute carriers in the regulation of innate antiviral responses, and they have potential implications for virus-host interactions and antiviral therapies. : Verrier et al. identify the sodium taurocholate co-transporter NTCP as a host factor regulating HCV infection. NTCP-mediated bile acid transport regulates innate responses targeting HCV infection. NTCP is a mediator of innate immunity and plays a role in the infection of multiple hepatotropic viruses. Keywords: NTCP, hepatitis C virus, solute carrier, innate immune responses, viral entry, host factor, signaling, antiviral therapy, hepatitis B virus, hepatitis D virus

With more than 71 million people chronically infected, hepatitis C virus (HCV) is one of the leading causes of liver disease and hepatocellular carcinoma. While efficient antiviral therapies have entered clinical standard of care, the development of a protective vaccine is still elusive. Recent studies have shown that the HCV life cycle is closely linked to lipid metabolism. HCV virions associate with hepatocyte-derived lipoproteins to form infectious hybrid particles that have been termed lipo-viro-particles. The close association with lipoproteins is not only critical for virus entry and assembly but also plays an important role during viral pathogenesis and for viral evasion from neutralizing antibodies. In this review, we summarize recent findings on the functional role of apolipoproteins for HCV entry and assembly. Furthermore, we highlight the impact of HCV–apolipoprotein interactions for evasion from neutralizing antibodies and discuss the consequences for antiviral therapy and vaccine design. Understanding these interactions offers novel strategies for the development of an urgently needed protective vaccine.

Chronic hepatitis C virus (HCV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC) which are leading indications of liver transplantation (LT). To date, there is no vaccine to prevent HCV infection and LT is invariably followed by infection of the liver graft. Within the past years, direct-acting antivirals (DAAs) have had a major impact on the management of chronic hepatitis C, which has become a curable disease in the majority of DAA-treated patients. In contrast to DAAs that target viral proteins, host-targeting agents (HTAs) interfere with cellular factors involved in the viral life cycle. By acting through a complementary mechanism of action and by exhibiting a generally higher barrier to resistance, HTAs offer a prospective option to prevent and treat viral resistance. Indeed, given their complementary mechanism of action, HTAs and DAAs can act in a synergistic manner to reduce viral loads. This review summarizes the different classes of HTAs against HCV infection that are in preclinical or clinical development and highlights their potential to prevent HCV infection, e.g., following LT, and to tailor combination treatments to cure chronic HCV infection.

Chronic hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases and hepatocellular carcinoma (HCC) worldwide. In the past few years, anti-HCV therapies have undergone a revolution with the approval of multiple direct-acting antivirals (DAAs), which enable interferon-free treatments with considerable improvement of sustained virologic response in patients. Today, DAAs have become the standard of care for HCV therapy. However, several limitations remain, which include access to therapy, treatment failure in a subset of patients and persistent risk of HCC development following cure in patients with advanced fibrosis. By targeting conserved host proteins involved in the HCV life cycle, host-targeting agents (HTAs) offer opportunities for pan-genotypic antiviral approaches with a high barrier to drug resistance. Moreover, when applied in combination with DAAs, HTAs could improve the management of difficult-to-treat patients by acting through a complementary mechanism of action. In this review, we summarize the different HTAs evaluated in preclinical and clinical development and discuss their potential role for anti-HCV therapies.

Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.

Hepatitis C virus (HCV) infection is a worldwide health problem and is one of the main causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Despite recent improvements, effective treatments for HCC are still missing and new tools for early detection are needed. Non-coding RNAs (ncRNAs) have emerged as important regulators of gene expression and key players in human carcinogenesis, including HCC. Aberrant expression of ncRNAs is associated with HCC metastasis, invasion, dissemination, and recurrence. This review will focus on the recent advances in ncRNA expression profiles, their dysregulation in HCV-related HCC, and the clinical perspective of ncRNA signatures for the early detection of HCC.

The precise role of adaptive immune responses in the clinical outcome of HCV infection is still only partially defined. Recent studies suggest that viral-host cell interactions during the acute phase of infection are essential for viral clearance or progression into chronic HCV infection. This review focuses on different aspects of the adaptive immune responses as determinants of the different outcomes of HCV infection, clearance or persistent infection, and outlines current concepts of HCV evasion strategies. Unravelling these important mechanisms of virus-host interaction will contribute to the development of novel strategies to prevent and control HCV infection.

Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma (HCC) making the virus the most common cause of liver failure and transplantation. HCV is estimated to chronically affect 130 million individuals and to lead to more than 350,000 deaths per year worldwide. A vaccine is currently not available. The recently developed direct acting antivirals (DAAs) have markedly increased the efficacy of the standard of care but are not efficient enough to completely cure all chronically infected patients and their toxicity limits their use in patients with advanced liver disease, co-morbidity or transplant recipients. Because of the host restriction, which is limited to humans and non-human primates, in vivo study of HCV infection has been hampered since its discovery more than 20 years ago. The chimpanzee remains the most physiological model to study the innate and adaptive immune responses, but its use is ethically difficult and is now very restricted and regulated. The development of a small animal model that allows robust HCV infection has been achieved using chimeric liver immunodeficient mice, which are therefore not suitable for studying the adaptive immune responses. Nevertheless, these models allowed to go deeply in the comprehension of virus-host interactions and to assess different therapeutic approaches. The immunocompetent mouse models that were recently established by genetic humanization have shown an interesting improvement concerning the study of the immune responses but are still limited by the absence of the complete robust life cycle of the virus. In this review, we will focus on the relevant available animal models of HCV infection and their usefulness for deciphering the HCV life cycle and virus-induced liver disease, as well as for the development and evaluation of new therapeutics. We will also discuss the perspectives on future immunocompetent mouse models and the hurdles to their development.

Chronic hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are major causes of liver disease and hepatocellular carcinoma worldwide. Despite the presence of an efficient preventive vaccine, more than 250 million patients are chronically infected with HBV. Current antivirals effectively control but only rarely cure chronic infection. While the molecular biology of the two viruses has been characterized in great detail, the absence of robust cell culture models for HBV and/or HDV infection has limited the investigation of virus-host interactions. Native hepatoma cell lines do not allow viral infection, and the culture of primary hepatocytes, the natural host cell for the viruses, implies a series of constraints restricting the possibilities of analyzing virus-host interactions. Recently, the discovery of the sodium taurocholate co-transporting polypeptide (NTCP) as a key HBV/HDV cell entry factor has opened the door to a new era of investigation, as NTCP-overexpressing hepatoma cells acquire susceptibility to HBV and HDV infections. In this review, we summarize the major cell culture models for HBV and HDV infection, discuss their advantages and limitations and highlight perspectives for future developments.

Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, first infect the liver for an initial round of replication before the emergence of pathogenic blood stages. Sporozoites represent attractive targets for antimalarial preventive strategies, yet the mechanisms of parasite entry into hepatocytes remain poorly understood. Here we show that the two main species causing malaria in humans, Plasmodium falciparum and Plasmodium vivax, rely on two distinct host cell surface proteins, CD81 and the Scavenger Receptor BI (SR-BI), respectively, to infect hepatocytes. By contrast, CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P. berghei. Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage. Our data provide molecular insights into the invasion pathways used by different malaria parasites to infect hepatocytes, and establish a functional link between a sporozoite putative ligand and host cell receptors.

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs.

Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies.Using genetic immunization, we produced four monoclonal antibodies (mAbs) against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines.The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity.A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

The poor response to the combined antiviral therapy of pegylated alfa-interferon and ribavarin for hepatitis C virus (HCV) infection may be linked to mutations in the viral envelope gene E1E2 (env), which can result in escape from the immune response and higher efficacy of viral entry. Mutations that result in failure of therapy most likely require compensatory mutations to achieve sufficient change in envelope structure and function. Compensatory mutations were investigated by determining positions in the E1E2 gene where amino acids (aa) covaried across groups of individuals. We assessed networks of covarying positions in E1E2 sequences that differentiated sustained virological response (SVR) from non-response (NR) in 43 genotype 1a (17 SVR), and 49 genotype 1b (25 SVR) chronically HCV-infected individuals. Binary integer programming over covariance networks was used to extract aa combinations that differed between response groups. Genotype 1a E1E2 sequences exhibited higher degrees of covariance and clustered into 3 main groups while 1b sequences exhibited no clustering. Between 5 and 9 aa pairs were required to separate SVR from NR in each genotype. aa in hypervariable region 1 were 6 times more likely than chance to occur in the optimal networks. The pair 531-626 (EI) appeared frequently in the optimal networks and was present in 6 of 9 NR in one of the 1a clusters. The most frequent pairs representing SVR were 431-481 (EE), 500-522 (QA) in 1a, and 407-434 (AQ) in 1b. Optimal networks based on covarying aa pairs in HCV envelope can indicate features that are associated with failure or success to antiviral therapy.

Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.