Hepatitis C virus (HCV) is a major cause of liver disease. Therapeutic options are limited and preventive strategies are absent. 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 co-factors for HCV entry. Blocking of kinase function by approved inhibitors broadly inhibited HCV infection of all major HCV genotypes and viral escape variants in cell culture and an animal model in vivo. Receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and membrane fusion. These results identify RTKs as novel HCV entry co-factors and uncover that kinase inhibitors have significant antiviral activity. Inhibition of RTK function may constitute a novel approach for prevention and treatment of HCV infection.

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.Enveloped viruses penetrate their host cells through a complex series of interactions between the viral surface and the cell membrane. This requires the attachment of the viral envelope glycoproteins to specific cell surface receptors and subsequent membrane fusion. Highly specific viral surface glycoproteins, named fusion proteins, catalyze the latter reaction by overcoming inherent energy barriers (10, 33). To date, two classes of virus fusion proteins have been defined (33): class I fusion proteins, the most well characterized of which is influenza virus hemagglutinin (HA) (62), and class II viral fusion proteins, exemplified by the E glycoprotein of tick-borne encephalitis virus (51), a flavivirus from the family Flaviviridae, and the E1 glycoprotein of Semliki Forest virus (26), an alphavirus from the Togaviridae family. Whereas their structural characteristics are markedly different, evidence suggests that class I and class II fusion proteins share an overall similar mechanism of membrane fusion (reviewed in reference 33). At an essential stage during fusion, the fusion protein bridges the gap between the viral and cell membranes by simultaneously interacting with them. The exposure and membrane insertion of a hydrophobic stretch of about 15 residues, called the "fusion peptides" or "fusion loops," mediate this crucial step (20, 54). For influenza virus HA and sev...

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.

No abstract

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. (HEPATOLOGY 2014;59:776-788) H epatitis C virus (HCV) cell entry is a multistep process that is mediated by virus surface components, such as HCV E1E2 envelope glycoproteins and viral-associated lipoproteins. 1 During the early steps of virus entry, the E1E2 complexes directly interact with some entry factors such as the scavenger receptor class B type 1 (SR-BI) 2 and the tetraspanin CD81. 3 The Claudin-1, 4 Occludin, 5 tight junction proteins, and the epidermal growth factor receptor (EGFR) 6 are also known to be critical for Abbreviations: HCV, hepatitis C virus; HCVcc, hepatitis C virus produced in cell culture; HCVpp, hepatitis C virus pseudoparticle; DMEM, Dulbecco's modified Eagle's medium; Mab, monoclonal antibody; PBS, phosphate-buffered saline; FCS, fetal calf serum; ffu, focus forming unit; SR-BI, scavenger receptor class B type I; sE2, soluble E2.From the

Background and aims. Liver macrophages can be both involved in 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 (PLM) and primary blood monocytes differentiated into pro-inflammatory or anti-inflammatory macrophages (M1-MDM or M2-MDM, respectively).Results. We evidenced the presence of HBc protein within macrophages in liver biopsies from HBV-infected patients and higher levels of anti-inflammatory macrophages markers, compared to non-infected ones. Ex vivo exposure of naive PLM to HBV led to a reduced secretion of proinflammatory cytokines. Upon exposure to HBV or HBV-producing cells during differentiation and activation, M1-MDM secreted less IL-6 and IL-1β, whereas M2-MDM secreted more IL-10 when exposed to HBV during activation. Finally, cytokines produced by M1-MDM, but not those produced by HBV-exposed M1-MDM, decreased HBV infection of hepatocytes.Conclusions. Altogether, our data strongly suggest that HBV modulates liver macrophage functions to favour its establishment. Lay summary: HBV modulates liver macrophages function in order to favour its establishment and likely its maintenance. It impairs the production of the antiviral cytokine IL-1β while promoting that of IL-10 in the microenvironment. This phenotype can be recapitulated in naive liver macrophages or monocytes-derived-macrophages ex vivo by short exposure to the virus or cells replicating the virus, thus suggesting an "easy to implement" mechanism of inhibition.

No abstract

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.