Summary Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5′UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. While xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin (OCLN) comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of SCARB1 for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as show that a recombinant vaccinia virus (rVV) vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.

Hepatitis C virus (HCV), which infects 2-3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation1. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough, and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays and/or terminal processing of potentially precious samples. Live-cell imaging avoids this, but necessitates genetically modified reporter viruses, which often exhibit profound replication defects. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.enveloped virus | hepacivirus | lipoviral particle | virus structure | virus assembly H epatitis C virus (HCV) is an important human pathogen that infects the liver and establishes chronic infection in the majority of cases, leading to cirrhosis and hepatocellular carcinoma (HCC) over the course of many years. More than 170 million people, ∼3% of the world's population, have been infected with HCV. Each year, 4-5% of patients with HCVinduced cirrhosis develop HCC, making HCV infection the leading indicator for liver transplantation in many areas of the world (1). Surgery, however, does not provide a cure because the donor organ universally becomes reinfected. A prophylactic vaccine is not available and despite the recent addition of HCV-specific protease inhibitors to the pegylated (peg)-IFN and ribavirin regimen, which has increased the cure rate, better therapies are still needed to solve the emergence of resistant variants, severe side effects and suboptimal response rates in cirrhotic patients (2).HCV is a single-stranded, positive-sense RNA virus in the family Flaviviridae. The HCV genome is ∼9.6 kb in length and encodes a long polyprotein of more than 3000 amino acids that is proteolytically processed to generate 10 mature viral proteins. Viral structural proteins are encoded by the first third of the polyprotein and include core or capsid protein (C) and the envelope glycoproteins E1 and E2. p7 (a viroporin) and nonstructural proteins, encoded by the C-terminal two-thirds of the polyprotein, pla...

More than 130 million people world-wide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry1 we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice2. Here, we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viremia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing for the first time evidence for the completion of the entire HCV life-cycle in inbred mice. This genetically humanized mouse model opens new opportunities to genetically dissect HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug and vaccine candidates.

With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.

The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformationdependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.Hepatitis C virus (HCV) is the major etiological agent of both community-acquired and posttransfusion non-A, non-B viral hepatitis. Approximately 80% of infected patients develop chronic hepatitis, among which 20% to 30% progress to liver cirrhosis and end-stage liver disease. Chronic infection correlates with an increased risk of hepatocellular carcinoma. Currently available therapies are limited to administration of pegylated alpha interferon in combination with ribavirin (27). Such treatment is expensive, is often unsuccessful, and carries the risk of significant side effects. Consequently, the development of novel therapeutic approaches against HCV remains a high-priority goal.HCV is an enveloped virus of the family Flaviviridae whose viral genome is a single-stranded, positive-sense RNA of approximately 9.6 kb that encodes a single polyprotein of 3,010 to 3,033 amino acids that is cleaved into nine mature proteins by a combination of host and viral peptidases (24). The predicted structural components comprise the core (C) (ϳ21 kDa) and two heavily N-glycosylated envelope glycoproteins, E1 (ϳ31 kDa) and E2 (ϳ70 kDa). Both E1 and E2 are believed to be type I transmembrane proteins, with N-terminal ectodomains and C-terminal hydrophobic anchors. HCV entry into target cells occurs after attachment to specific cellular receptors via its surface glycoprotei...

Scavenger receptor class B type I (SR-BI) is an essential receptor for hepatitis C virus (HCV) and a cellHepatitis C virus (HCV) is a global blood-borne pathogen, with 3% of the world's population chronically infected. Most infections are asymptomatic, yet 60 to 80% become persistent and lead to severe fibrosis and cirrhosis, hepatic failure, or hepatocellular carcinoma (3). Currently available therapies are limited to the administration of pegylated alpha interferon in combination with ribavirin, which are expensive and often unsuccessful, with significant side effects (23, 36). Thus, the development of novel therapeutic approaches against HCV remains a high priority (18,40,60). Targeting the early steps of HCV infection may represent one such option, and much effort is being devoted to uncovering the mechanism of viral attachment and entry.The current view is that HCV entry into target cells occurs after attachment to specific cellular receptors via its surface glycoproteins E1 and E2 (27). The molecules to which HCV initially binds might constitute a diverse collection of cellular proteins, carbohydrates, and lipids that concentrate viruses on the cell surface and determine to a large extent which cell types, tissues, and organisms HCV can infect.CD81, claudin 1 (CLDN1), occludin (OCLN), and scavenger receptor class B type I (SR-BI) were previously shown to play essential roles in HCV cell entry (15,22,26,35,42,43,50,63,64).Recent reports suggest that CD81 engagement triggers intracellular signaling responses, ultimately leading to actin remodeling and the relocalization of CD81 to tight junctions (TJ) (11). Thus, CD81 may function as a bridge between the initial interaction of the virus with receptors on the basolateral surface of the hepatocyte and the TJ where two of the HCV entry molecules, CLDN1 and OCLN, are located. CD81 acts as a postbinding factor, and the TJ proteins CLDN1 and OCLN seem to be involved in late steps of HCV entry, such as HCV glycoprotein-dependent cell fusion (9,11,22). The discovery of TJ proteins as entry factors has added complexity to the model of HCV entry, suggesting parallels with other viruses like coxsackievirus B infection, where an initial interaction of the viral particle with the primary receptor decay-accelerating factor induces the lateral movement of the virus from the luminal surface to TJ, where coxsackievirus B binds coxsackievirusadenovirus receptor and internalization takes place (17).Much less is known about the specific role of SR-BI in virus * Corresponding author. Mailing address: Okairòs, via dei Castelli Romani 22,