Use of non-small cell lung cancer multicellular tumor spheroids to study the impact of chemotherapy.

BACKGROUND: Lung cancers represent the main cause of cancer related-death worldwide. Recently, immunotherapy alone or in combination with chemotherapy has deeply impacted the therapeutic care leading to an improved overall survival. However, relapse will finally occur, with no efficient second line treatment so far. New therapies development based on the comprehension of resistance mechanisms is necessary. However, the difficulties to obtain tumor samples before and after first line treatment hamper to clearly understand the consequence of these molecules on tumor cells and also to identify adapted second line therapies. METHODS: To overcome this difficulty, we developed multicellular tumor spheroids (MCTS) using characterized Non-Small Cell Lung Cancer (NSCLC) cell lines, monocytes from healthy donors and fibroblasts. MCTS were treated with carboplatin-paclitaxel or -gemcitabine combinations according to clinical administration schedules. The treatments impact was studied using cell viability assay, histological analyses, 3'RNA sequencing, real-time PCR, flow cytometry and confocal microscopy. RESULTS: We showed that treatments induced a decrease in cell viability and strong modifications in the transcriptomic profile notably at the level of pathways involved in DNA damage repair and cell cycle. Interestingly, we also observed a modification of genes expression considered as hallmarks of response to immune check point inhibitors and immunogenicity, particularly an increase in CD274 gene expression, coding for PD-L1. This result was validated at the protein level and shown to be restricted to tumor cells on MCTS containing fibroblasts and macrophages. This increase was also observed in an additional cell line, expressing low basal CD274 level. CONCLUSIONS: This study shows that MCTS are interesting models to study the impact of first line therapies using conditions close to clinical practice and also to identify more adapted second line or concomitant therapies for lung cancer treatment.

Specific Targeting of Mesothelin-Expressing Malignant Cells Using Nanobody-Functionalized Magneto-Fluorescent Nanoassemblies.

Introduction: Most current anti-cancer therapies are associated with major side effects due to a lack of tumor specificity. Appropriate vectorization of drugs using engineered nanovectors is known to increase local concentration of therapeutic molecules in tumors while minimizing their side effects. Mesothelin (MSLN) is a well-known tumor associated antigen overexpressed in many malignancies, in particular in malignant pleural mesothelioma (MPM), and various MSLN-targeting anticancer therapies are currently evaluated in preclinical and clinical assays. In this study, we described, for the first time, the functionalization of fluorescent organic nanoassemblies (NA) with a nanobody (Nb) targeting MSLN for the specific targeting of MSLN expressing MPM cancer cells. Methods: Cell lines from different cancer origin expressing or not MSLN were used. An Nb directed against MSLN was coupled to fluorescent NA using click chemistry. A panel of endocytosis inhibitors was used to study targeted NA internalization by cells. Cancer cells were grown in 2D or 3D and under a flow to evaluate the specificity of the targeted NA. Binding and internalization of the targeted NA were studied using flow cytometry, confocal microscopy and transmission electron microscopy. Results: We show that the targeted NA specifically bind to MSLN-expressing tumor cells. Moreover, such functionalized NA appear to be internalized more rapidly and in significantly larger proportions compared to naked ones in MSLN+ MPM cells, thereby demonstrating both the functionality and interest of the active targeting strategy. We demonstrated that targeted NA are mainly internalized through a clathrin-independent/dynamin-dependent endocytosis pathway and are directed to lysosomes for degradation. A 3D cell culture model based on MSLN-expressing multicellular tumor spheroids reveals NA penetration in the first superficial layers. Conclusion: Altogether, these results open the path to novel anticancer strategies based on MSLN-activated internalization of NA incorporating drugs to promote specific accumulation of active treatments in tumors.

ChemR23 activation reprograms macrophages toward a less inflammatory phenotype and dampens carcinoma progression.

Introduction: Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Methods: Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. Results: We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. Conclusion: These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.

HIRA Supports Hepatitis B Virus Minichromosome Establishment and Transcriptional Activity in Infected Hepatocytes.

BACKGROUND & AIMS: Upon hepatitis B virus (HBV) infection, partially double-stranded viral DNA converts into a covalently closed circular chromatinized episomal structure (cccDNA). This form represents the long-lived genomic reservoir responsible for viral persistence in the infected liver. Although the involvement of host cell DNA damage response in cccDNA formation has been established, this work investigated the yet-to-be-identified histone dynamics on cccDNA during early phases of infection in human hepatocytes. METHODS: Detailed studies of host chromatin-associated factors were performed in cell culture models of natural infection (ie, Na+-taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells, HepG2hNTCP) and primary human hepatocytes infected with HBV, by cccDNA-specific chromatin immunoprecipitation and loss-of-function experiments during early kinetics of viral minichromosome establishment and onset of viral transcription. RESULTS: Our results show that cccDNA formation requires the deposition of the histone variant H3.3 via the histone regulator A (HIRA)-dependent pathway. This occurs simultaneously with repair of the cccDNA precursor and independently from de novo viral protein expression. Moreover, H3.3 in its S31 phosphorylated form appears to be the preferential H3 variant found on transcriptionally active cccDNA in infected cultured cells and human livers. HIRA depletion after cccDNA pool establishment showed that HIRA recruitment is required for viral transcription and RNA production. CONCLUSIONS: Altogether, we show a crucial role for HIRA in the interplay between HBV genome and host cellular machinery to ensure the formation and active transcription of the viral minichromosome in infected hepatocytes.

Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B.

Hepatitis B virus-induced modulation of liver macrophage function promotes hepatocyte infection.

BACKGROUND & AIMS: Liver macrophages can be involved in both pathogen clearance and/or pathogenesis. To get further insight on their role during chronic hepatitis B virus (HBV) infections, our aim was to phenotypically and functionally characterize in vivo and ex vivo the interplay between HBV, primary human liver macrophages (PLMs) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDMs or M2-MDMs, respectively). METHODS: PLMs or primary blood monocytes, either ex vivo differentiated into M1-MDMs or M2-MDMs, were exposed to HBV and their activation followed by ELISA or quantitative reverse transcription PCR (RT-qPCR). Liver biopsies from HBV-infected patients were analysed by RT-qPCR or immunohistochemistry. Viral parameters in HBV-infected primary human hepatocytes and differentiated HepaRG cells were followed by ELISA, qPCR and RT-qPCR analyses. RESULTS: HBc protein was present within the macrophages of liver biopsies taken from HBV-infected patients. Macrophages from HBV-infected patients also expressed higher levels of anti-inflammatory macrophage markers than those from non-infected patients. Ex vivo exposure of naive PLMs to HBV led to reduced secretion of pro-inflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDMs secreted less IL-6 and IL-1ß, whereas M2-MDMs secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDMs, but not those produced by HBV-exposed M1-MDMs, decreased HBV infection of hepatocytes. CONCLUSIONS: Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour the establishment of infection. LAY SUMMARY: Hepatitis B virus modulates liver macrophage function in order to favour the establishment and likely maintenance of infection. It impairs the production of the antiviral cytokine IL-1ß, while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocyte-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition.

Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.

BACKGROUND & AIMS: Hepatitis B virus (HBV) persistence and the pathobiology of chronic HBV (CHB) infections result from the interplay between viral replication and host immune responses. We aimed to comprehensively analyse the expression of intrahepatic host genes as well as serum and liver HBV markers in a large cohort of untreated CHB patients. METHODS: One-hundred and five CHB patients untreated at the time of liver biopsy (34 HBeAg[+] and 71 HBeAg[-]) were analysed for the intrahepatic expression profile of 67 genes belonging to multiple innate immunity pathways. Results were correlated to serological (quantification of HBsAg [qHBsAg] and HBV DNA) and intrahepatic viral markers (total HBV DNA, pre-genomic RNA and covalently closed circular HBV DNA). RESULTS: Intrahepatic gene expression profiling revealed a strong downregulation of antiviral effectors, interferon stimulated genes, Toll-like and pathogen recognition receptor pathways in CHB patients as compared to non-infected controls, which was not directly correlated to HBV replication. A subset of genes [CXCL10, GBP1, IFITM1, IFNB1, IL10, IL6, ISG15, TLR3, SOCS1, SOCS3] was more repressed in HBeAg(-) respect to HBeAg(+) patients (median of serum HBV DNA 7.9×103vs. 7.9×107IU/ml, respectively). Notably, HBeAg(-) patients with lower qHBsAg (<5×103IU/ml) showed a relief of repression of genes belonging to multiple pathways. CONCLUSIONS: Our results show a strong impairment of innate immune responses in the liver of CHB patients. The association of low levels of qHBsAg with gene repression, if confirmed, might prove useful for the identification of patients who would most benefit from immune-modulators and/or HBsAg targeting agents as strategies to restore immune responsiveness. LAY SUMMARY: Chronic hepatitis B virus (HBV) infections represent a major public health problem worldwide. Over 200 million people are chronically infected and at risk of developing chronic hepatitis, liver cirrhosis and cancer. Our work aimed to understand the molecular consequences of chronic hepatitis B in the infected liver. It was conducted in a large cohort of untreated chronically infected HBV patients and analysed the expression of immunity and liver disease-related genes in the liver, with respect to markers of viral replication and persistence. Our results indicate that chronic HBV infection has a suppressive effect on immune responses, which was more pronounced with high levels of hepatitis B virus surface antigen (HBsAg). These data provide novel insight into the mechanisms of HBV persistence in the liver and suggest that approaches aimed at reducing HBsAg levels, may restore immune responsiveness against the virus.

Development of hepatocellular carcinoma in chronic hepatitis B patients with advanced fibrosis is independent of viral genotype.

Hepatitis B is leading cause of liver related morbidity in Asia with predominant genotypes B and C in East-Asia. Data on Serum, intrahepatic viral-markers, and long-term follow-up of prevalent genotypes (GT) B and C in patients with biopsy proven advanced fibrosis are sparse. To compare serum, intrahepatic viral-markers and development of hepatocellular carcinoma (HCC) in GT-B and C in patients with advanced fibrosis (Ishak ≥ 4). Sixty-three treatment-naïve patients identified with advanced fibrosis on liver-biopsy performed between 1998 and 2000 at Singapore General Hospital. FFPE tissue was available for 59 patients and serum for 42 patients. HBV-DNA was quantified in serum and liver while qHBsAg quantified in serum. Patients were followed-up till December 2015. The median age was 47 ± 16 years, with 77.7% males. About 19 were GT-B, 43 patients were GT-C, and 1 had both GT-B and C. Mean follow-up was 13.5 years. The median serum HBV-DNA was 6.25 ± 2.17 and 6.58 ± 1.85 log IU/ml, serum HBsAg was 3.29 ± 0.80 and 3.45 ± 1.85 log IU/ml, and intrahepatic HBV-DNA was 0.52 ± 3.73 copies/cell and 0.4 ± 1.37 copies/cell in the GT-B and C, respectively (P > 0.1 in all). Complete cirrhosis (Ishak-6) was present in 47.6%, Ishak-5 fibrosis in 33.3%, and Ishak-4 fibrosis in 19% at recruitment. On follow-up HCC developed in 8/43 in GT-C and in 3/19 GT-B (P = 0.86). Advanced age and cirrhosis were significant factors for development of HCC. No difference in serum HBV-DNA, qHBsAg or intrahepatic HBV-DNA was seen in the two genotypes. HCC development seen over long-term follow-up was independent of genotypes in patients with advanced fibrosis. J. Med. Virol. 89:845-848, 2017. © 2016 Wiley Periodicals, Inc.

Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.

In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were type-I IFNs, IFNγ, type-III IFNs, TNFα, IL-6, IL-1ß, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1ß, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1ß and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1ß was found to have a very potent antiviral effect against HBV in vitro. HBV was previously shown to promptly inhibit IL-1ß production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1ß may help to further inhibit HBV replication in vivo.

Specialization of Hepatitis C Virus Envelope Glycoproteins for B Lymphocytes in Chronically Infected Patients.

UNLABELLED: Hepatitis C virus (HCV) productively infects hepatocytes. Virion surface glycoproteins E1 and E2 play a major role in this restricted cell tropism by mediating virus entry into particular cell types. However, several pieces of evidence have suggested the ability of patient-derived HCV particles to infect peripheral blood mononuclear cells. The viral determinants and mechanisms mediating such events remain poorly understood. Here, we aimed at isolating viral determinants of HCV entry into B lymphocytes. For this purpose, we constructed a library of full E1E2 sequences isolated from serum and B lymphocytes of four chronically infected patients. We observed a strong phylogenetic compartmentalization of E1E2 sequences isolated from B lymphocytes in one patient, indicating that E1E2 glycoproteins can represent important mediators of the strong segregation of two specialized populations in some patients. Most of the E1E2 envelope glycoproteins were functional and allowed transduction of hepatocyte cell lines using HCV-derived pseudoparticles. Strikingly, introduction of envelope glycoproteins isolated from B lymphocytes into the HCV JFH-1 replicating virus switched the entry tropism of this nonlymphotropic virus from hepatotropism to lymphotropism. Significant detection of viral RNA and viral proteins within B cells was restricted to infections with JFH-1 harboring E1E2 from lymphocytes and depended on an endocytic, pH-dependent entry pathway. Here, we achieved for the first time the isolation of HCV viral proteins carrying entry-related lymphotropism determinants. The identification of genetic determinants within E1E2 represents a first step for a better understanding of the complex relationship between HCV infection, viral persistence, and extrahepatic disorders. IMPORTANCE: Hepatitis C virus (HCV) mainly replicates within the liver. However, it has been shown that patient-derived HCV particles can slightly infect lymphocytes in vitro and in vivo, highlighting the existence of lymphotropism determinants within HCV viral proteins. We isolated HCV envelope glycoproteins from patient B lymphocytes that conferred to a nonlymphotropic HCV the ability to enter B cells, thus providing a platform for characterization of HCV entry into lymphocytes. This unusual tropism was accompanied by a loss of entry function into hepatocytes, suggesting that HCV lymphotropic variants likely constitute a distinct but parallel source for viral persistence and immune escape within chronically infected patients. Moreover, the level of genetic divergence of B-cell-derived envelopes correlated with their degree of lymphotropism, underlining a long-term specialization of some viral populations for B-lymphocytes. Consequently, the clearance of both hepatotropic and nonhepatotropic HCV populations may be important for effective treatment of chronically infected patients.

Ribavirin restores IFNα responsiveness in HCV-infected livers by epigenetic remodelling at interferon stimulated genes.

OBJECTIVES: Caveats in the understanding of ribavirin (RBV) mechanisms of action has somehow prevented the development of better analogues able to further improve its therapeutic contribution in interferon (IFN)-based and direct antiviral agent-based regimens for chronic HCV or other indications. Here, we describe a new mechanism by which RBV modulates IFN-stimulated genes (ISGs) and contributes to restore hepatic immune responsiveness. DESIGN: RBV effect on ISG expression was monitored in vitro and in vivo, that is, in non-transformed hepatocytes and in the liver of RBV mono-treated patients, respectively. Modulation of histone modifications and recruitment of histone-modifying enzymes at target promoters was analysed by chromatin immunoprecipitation in RBV-treated primary human hepatocytes and in patients' liver biopsies. RESULTS: RBV decreases the mRNA levels of several abnormally preactivated ISGs in patients with HCV, who are non-responders to IFN therapy. RBV increases G9a histone methyltransferase recruitment and histone-H3 lysine-9 dimethylation/trimethylation at selected ISG promoters in vitro and in vivo. G9a pharmacological blockade abolishes RBV-induced ISG downregulation and severely impairs RBV ability to potentiate IFN antiviral action and induction of ISGs following HCV infection of primary human hepatocytes. CONCLUSIONS: RBV-induced epigenetic changes, leading to decreased ISG expression, restore an IFN-responsive hepatic environment in patients with HCV, which may also prove useful in IFN-free regimens.

Determinants Involved in Hepatitis C Virus and GB Virus B Primate Host Restriction.

UNLABELLED: Hepatitis C virus (HCV) only infects humans and chimpanzees, while GB virus B (GBV-B), another hepatotropic hepacivirus, infects small New World primates (tamarins and marmosets). In an effort to develop an immunocompetent small primate model for HCV infection to study HCV pathogenesis and vaccine approaches, we investigated the HCV life cycle step(s) that may be restricted in small primate hepatocytes. First, we found that replication-competent, genome-length chimeric HCV RNAs encoding GBV-B structural proteins in place of equivalent HCV sequences designed to allow entry into simian hepatocytes failed to induce viremia in tamarins following intrahepatic inoculation, nor did they lead to progeny virus in permissive, transfected human Huh7.5 hepatoma cells upon serial passage. This likely reflected the disruption of interactions between distantly related structural and nonstructural proteins that are essential for virion production, whereas such cross talk could be restored in similarly designed HCV intergenotypic recombinants via adaptive mutations in NS3 protease or helicase domains. Next, HCV entry into small primate hepatocytes was examined directly using HCV-pseudotyped retroviral particles (HCV-pp). HCV-pp efficiently infected tamarin hepatic cell lines and primary marmoset hepatocyte cultures through the use of the simian CD81 ortholog as a coreceptor, indicating that HCV entry is not restricted in small New World primate hepatocytes. Furthermore, we observed genomic replication and modest virus secretion following infection of primary marmoset hepatocyte cultures with a highly cell culture-adapted HCV strain. Thus, HCV can successfully complete its life cycle in primary simian hepatocytes, suggesting the possibility of adapting some HCV strains to small primate hosts. IMPORTANCE: Hepatitis C virus (HCV) is an important human pathogen that infects over 150 million individuals worldwide and leads to chronic liver disease. The lack of a small animal model for this infection impedes the development of a preventive vaccine and pathogenesis studies. In seeking to establish a small primate model for HCV, we first attempted to generate recombinants between HCV and GB virus B (GBV-B), a hepacivirus that infects small New World primates (tamarins and marmosets). This approach revealed that the genetic distance between these hepaciviruses likely prevented virus morphogenesis. We next showed that HCV pseudoparticles were able to infect tamarin or marmoset hepatocytes efficiently, demonstrating that there was no restriction in HCV entry into these simian cells. Furthermore, we found that a highly cell culture-adapted HCV strain was able to achieve a complete viral cycle in primary marmoset hepatocyte cultures, providing a promising basis for further HCV adaptation to small primate hosts.

Low cross-neutralization of hepatitis C correlates with liver disease in immunocompromized patients.

BACKGROUND AND AIMS: Chronic hepatitis C virus (HCV) infection causes severe liver disease in HIV-infected patients and liver transplant recipients. The impact of serum and immunoglobulin on viral entry was analysed in these patients. METHOD: Sera from 60 anti-HCV positive patients, including 30 who were also anti-HIV positive, were tested with HCVpp from different genotypes (1a, 1b, 3 and 4) and with HCVcc (H77/JFH1). Seventeen HIV-seropositive and 13 HIV-seronegative patients with decompensated liver disease were studied before and after liver transplant. RESULTS: Serum neutralization was markedly lower after liver transplant and in HIV patients than in mono-infected immune-competent individuals. This effect was due to low antibody-mediated neutralization. In HIV patients, low neutralization was correlated with low lymphocyte T CD4 cell counts and the severity of liver disease. To characterize neutralization, we tested HCVpp lacking hypervariable region (HVR1) and SR-BI receptor cholesterol transfer inhibition by BLT-4. These experiments showed that neutralization was strongly dependent on the HVR1 and the SR-BI receptor. HVR1 sequences showed that selective pressures were low in immune-compromised patients and highly correlated to HCV neutralization after liver transplant. Neutralization experiments were reproduced with HCV strain JFH1. CONCLUSION: Serum neutralization in HIV-coinfected patients and HCV-infected liver transplant recipients is poor enhancing HCV entry through HVR1/SR-BI interplay. This may contribute to the severity of hepatitis C in these settings.

Critical interaction between E1 and E2 glycoproteins determines binding and fusion properties of hepatitis C virus during cell entry.

UNLABELLED: Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 are important mediators for productive cell entry. However, knowledge about their structure, intra- or intermolecular dialogs, and conformational changes is scarce, limiting the design of therapeutic strategies targeting E1E2. Here we sought to investigate how certain domains of E1 and E2 have coevolved to optimize their interactions to promote efficient HCV entry. For this purpose we generated chimeric E1E2 heterodimers derived from two HCV 1a strains to identify and characterize crosstalk between their domains. We found an E1E2 combination that drastically impaired the infectivity of cell culture-derived HCV particles, whereas the reciprocal E1E2 combination led to increased infectivity. Using HCV pseudoparticle assays, we confirmed the opposing entry phenotypes of these heterodimers. By mutagenesis analysis, we identified a particular crosstalk between three amino acids of E1 and the domain III of E2. Its modulation leads to either a full restoration of the functionality of the suboptimal heterodimer or a destabilization of the functional heterodimer. Interestingly, we found that this crosstalk modulates E1E2 binding to HCV entry receptors SR-BI and CD81. In addition, we found for the first time that E1E2 complexes can interact with the first extracellular loop of Claudin-1, whereas soluble E2 did not. These results highlight the critical role of E1 in the modulation of HCV binding to receptors. Finally, we demonstrated that this crosstalk is involved in membrane fusion. CONCLUSIONS: These results reveal a multifunctional and crucial interaction between E1 and E2 for HCV entry into cells. Our study highlights the role of E1 as a modulator of HCV binding to receptors and membrane fusion, underlining its potential as an antiviral target.

Identification of interactions in the E1E2 heterodimer of hepatitis C virus important for cell entry.

Several conserved domains critical for E1E2 assembly and hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains involved in cross-talk between either glycoprotein must be defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains and to identify their functional partners, we analyzed a set of intergenotypic E1E2 heterodimers derived from E1 and E2 of different genotypes. The infectivity of virions indicated that Con1 E1 did not form functional heterodimers when associated with E2 from H77. Biochemical analyses demonstrated that the reduced infectivity was not related to alteration of conformation and incorporation of Con1 E1/H77 E2 heterodimers but rather to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in order to restore functional heterodimers. We found that both the ectodomain and transmembrane domain of E1 influenced infectivity. Site-directed mutagenesis highlighted the role of amino acids 359, 373, and 375 in transmembrane domain in entry. In addition, we identified one domain involved in entry within the N-terminal part of E1, and we isolated a motif at position 219 that is critical for H77 function. Interestingly, using additional chimeric E1E2 complexes harboring substitutions in this motif, we found that the transmembrane domain of E1 acts as a partner of this motif. Therefore, we characterized domains of E1 and E2 that have co-evolved inside a given genotype to optimize their interactions and allow efficient entry.

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.

Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus.

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.