-Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10 5 infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-a and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, at least one factor critical to this process remains unknown (reviewed in refs 1-3). Using an iterative expression cloning approach we identified claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, as essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Moreover, antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of this inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells1,2, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp)3 or HCV produced in cell culture (HCVcc)(unpublished data), suggesting a block at the level of entry. Through an iterative cDNA library screening approach we have identified human occludin (OCLN) as an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for HCV-susceptibility of human cells, since its overexpression in uninfectable cells specifically enhanced HCVpp uptake while its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors, CD814, scavenger receptor class B type I (SR-BI)5, and claudin-1 (CLDN1)6. While the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins for promoting HCV entry; both OCLN and CD81, however, must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process and provides a major advance towards efforts to develop small animal models for HCV.

Summary The ongoing epidemic of Zika virus (ZIKV) illustrates the importance of flaviviruses as emerging human pathogens. All vector-borne flaviviruses studied thus far have to overcome type I interferon (IFN) to replicate and cause disease in vertebrates. The mechanism(s) by which ZIKV antagonizes IFN signaling is unknown. Here, we report that the nonstructural protein NS5 of ZIKV and other flaviviruses examined could suppress IFN signaling, but through different mechanisms. ZIKV NS5 expression resulted in proteasomal degradation of the IFN-regulated transcriptional activator STAT2 from humans but not mice, which may explain the requirement for IFN-deficiency to observe ZIKV-induced disease in mice. The mechanism of ZIKV NS5 resembles dengue virus (DENV) NS5 and not its closer relative, Spondweni virus (SPOV). However, unlike DENV, ZIKV did not require the E3 ubiquitin ligase UBR4 to induce STAT2 degradation. Hence, flavivirus NS5 proteins exhibit a remarkable functional convergence in IFN antagonism, albeit by virus-specific mechanisms.

SUMMARY The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A-methyltransferases or an m6A-demethylase respectively increases and decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determine that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Together, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.

We introduce two large-scale resources for functional analysis of microRNA—a decoy/sponge library for inhibiting microRNA function and a sensor library for monitoring microRNA activity. To take advantage of the sensor library, we developed a high-throughput assay called Sensor-seq, which permits the activity of hundreds of microRNAs to be quantified simultaneously. Using this approach, we show that only the most abundant microRNAs within a cell mediate significant target suppression. Over 60% of detected microRNAs had no discernible activity, indicating that the functional ‘miRNome’ of a cell is considerably smaller than currently inferred from profiling studies. Moreover, some highly expressed microRNAs exhibit relatively weak activity, which in some cases correlated with a high target-to-microRNA ratio or increased nuclear localization of the microRNA. Finally, we show that the microRNA decoy library can be used for pooled loss-of-function studies. These tools provide valuable resources for studying microRNA biology and for microRNA-based therapeutics.

Hepatitis C virus (HCV) is an important human pathogen associated with chronic liver disease. Recently, based on a genotype 2a isolate, tissue culture systems supporting complete replication and infectious virus production have been developed. In this study, we used cell culture-produced infectious HCV to analyze the viral entry pathway into Huh-7.5 cells. Bafilomycin A1 and concanamycin A, inhibitors of vacuolar ATPases, prevented HCV entry when they were present prior to infection and had minimal effect on downstream replication events. HCV entry therefore appears to be pH dependent, requiring an acidified intracellular compartment. For many other enveloped viruses, acidic pH triggers an irreversible conformational change, which promotes virion-endosomal membrane fusion. Such viruses are often inactivated by low pH. In the case of HCV, exposure of virions to acidic pH followed by return to neutral pH did not affect their infectivity. This parallels the observation made for the related pestivirus bovine viral diarrhea virus. Low pH could activate the entry of cell surface-bound HCV but only after prolonged incubation at 37°C. This suggests that there are rate-limiting, postbinding events that are needed to render HCV competent for low-pH-triggered entry. Such events may involve interaction with a cellular coreceptor or other factors but do not require cathepsins B and L, late endosomal proteases that activate Ebola virus and reovirus for entry.Hepatitis C virus (HCV) is the sole member of the Hepacivirus genus within the family Flaviviridae. HCV can cause a persistent infection in humans that is associated with chronic liver disease and hepatocellular carcinoma (27). Flaviviridae are enveloped viruses with a single-stranded RNA genome of positive polarity (reviewed in reference 24). Within the virion, HCV genomic RNA is complexed with multiple copies of capsid protein (C). The viral envelope bears on its surface two type I integral membrane envelope glycoproteins, E1 and E2, which form heterodimers (30). Both E1 and E2 have been shown to accumulate in the endoplasmic reticulum, where particles are thought to assemble (30).Infection with an enveloped virus requires a fusion event between the viral membrane and a cellular membrane. This event can occur at the cell surface, as demonstrated by human immunodeficiency virus (HIV) and herpes simplex virus (HSV), where binding to one or more receptors induces conformational changes in the envelope glycoprotein, allowing membrane fusion at neutral pH. Alternatively, the fusion event can occur within an endosomal compartment in the presence of low pH (reviewed in reference 39), as has been previously described for classical flaviviruses and pestiviruses as well as alphaviruses (8,12,14,15,20).Previous studies of HCV entry have been based on retroviral pseudotypes bearing HCV E1 and E2 glycoproteins (HCVpp) (3,17). Such pseudotypes were shown to undergo pH-dependent entry into Huh-7 cells in a CD81-dependent manner and could be neutralized by certain anti-E2 monoclonal an...