Solute Carrier NTCP Regulates Innate Antiviral Immune Responses Targeting Hepatitis C Virus Infection of Hepatocytes.

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.

Use of a Closed Culture System to Improve the Safety of Lentiviral Vector Production.

We evaluated the possibility of introducing a combination of six oncogenes into primary porcine hepatocytes (PPH) using a lentiviral vector (LV)-mediated gene transfer in order to develop a porcine hepatocellular carcinoma model based on autologous transplantation of ex vivo-transformed hepatocytes. The six oncogenes were introduced into three plasmids, hence enabling the production of LVs encoding a luciferase reporter gene and hTERT+p53(DD), cyclinD1+CDK4(R24C), and c-myc(T58A)+HRas(G21V) genes, respectively. In order to improve the protection of the laboratory personnel manipulating such LVs, we used a compact cell culture cassette (CliniCell(®) device) as a closed cell culture system. We demonstrated that the CliniCell device allows to produce LVs, through plasmid transfection of 293T cells, and, after transfer to a second cassette, to transduce PPH with a similar efficacy as conventional open cell culture systems such as flasks or Petri dishes. Additionally, it is possible to cryopreserve at -80°C the transduced cells, directly in the CliniCell device used for the transduction. In conclusion, the use of a closed culture system for the safe handling of oncogene-encoding LVs lays the foundation for the development of porcine tumor models based on the autologous transplantation of ex vivo-transformed primary cells.

CD81-receptor associations--impact for hepatitis C virus entry and antiviral 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.

Functional analysis of claudin-6 and claudin-9 as entry factors for hepatitis C virus infection of human hepatocytes by using monoclonal antibodies.

The relevance of claudin-6 and claudin-9 in hepatitis C virus (HCV) entry remains elusive. We produced claudin-6- or claudin-9-specific monoclonal antibodies that inhibit HCV entry into nonhepatic cells expressing exogenous claudin-6 or claudin-9. These antibodies had no effect on HCV infection of hepatoma cells or primary hepatocytes. Thus, although claudin-6 and claudin-9 can serve as entry factors in cell lines, HCV infection into human hepatocytes is not dependent on claudin-6 and claudin-9.

A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

BACKGROUND AND AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha.

UNLABELLED: Interferon-alpha (IFN-α) exhibits its antiviral activity through signal transducer and activator of transcription protein (STAT) signaling and the expression of IFN response genes (IRGs). Viral infection has been shown to result in activation of epidermal growth factor receptor (EGFR)-a host cell entry factor used by several viruses, including hepatitis C virus. However, the effect of EGFR activation for cellular antiviral responses is unknown. Here, we uncover cross-talk between EGFR and IFN-α signaling that has a therapeutic effect on IFN-α-based therapies and functional relevance for viral evasion and IFN resistance. We show that combining IFN-α with the EGFR inhibitor, erlotinib, potentiates the antiviral effect of each compound in a highly synergistic manner. The extent of the synergy correlated with reduced STAT3 phosphorylation in the presence of erlotinib, whereas STAT1 phosphorylation was not affected. Furthermore, reduced STAT3 phosphorylation correlated with enhanced expression of suppressors of cytokine signaling 3 (SOCS3) in the presence of erlotinib and enhanced expression of the IRGs, radical S-adenosyl methionine domain containing 2 and myxovirus resistance protein 1. Moreover, EGFR stimulation reduced STAT1 dimerization, but not phosphorylation, indicating that EGFR cross-talk with IFN signaling acts on the STATs at the level of binding DNA. CONCLUSIONS: Our results support a model where inhibition of EGFR signaling impairs STAT3 phosphorylation, leading to enhanced IRG expression and antiviral activity. These data uncover a novel role of EGFR signaling in the antiviral activity of IFN-α and open new avenues of improving the efficacy of IFN-α-based antiviral therapies.

HRas signal transduction promotes hepatitis C virus cell entry by triggering assembly of the host tetraspanin receptor complex.

Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin ß1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.

Hepatitis C virus internalization.

Hepatitis C virus (HCV) infection is a major cause of fibrosis, cirrhosis and hepatocellular carcinoma worldwide and thus an important public health problem. No vaccine is available and the current treatment does not cure all patients. A better understanding of the HCV life cycle will aid in developing new antivirals. HCV entry is defined as the sequence of events initiated by attachment of the virus to its host cell end leading to the release of the viral genome into the cytoplasm. HCV entry into hepatocytes is a highly elaborated process involving many viral and cellular factors. Various cellular factors expressed on the surface of hepatocytes contribute to the early stages of viral attachment and entry. In addition, various intracellular proteins also play an important role during virus internalization. This review focuses on recent findings on the HCV entry process and highlights the factors and signaling pathways involved.

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.