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.

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77. Hepatitis C virus (HCV) is a blood-borne hepatotropic virus that infects ϳ170 million people worldwide. Approximately 70% of infected individuals progress to chronic liver disease, which carries an increased risk of cirrhosis and hepatocellular carcinoma (7). In general, treatment of chronic HCV infection is complicated by resistance due to extensive genetic diversity. HCV has been classified into seven major genotypes, which differ by ϳ30% at the nucleotide level (4), and this positivesense, single-stranded RNA virus has a capacity for rapid evolution of variant viruses during persistent infection. The current treatment, pegylated ␣ 2a interferon (IFN-␣ 2a ) and ribavirin, has variable side effects and response rates depending on the virus and host genotype (16). No vaccine is currently available, and preclinical development has been hampered by a lack of understanding of which conserved epitopes on the HCV structural proteins should be targeted.HCV contains an ϳ9.6-kb RNA genome that is translated as a single polyprotein and then cleaved by viral and host proteases into structural proteins (core, E1, and E2), p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (39). Viral attachment and entry are mediated by the envelope glycoproteins, E1 and E2. Four attachment or entry receptors that are required for infection of hepatocytes have been identified, including CD81 (53), scavenger receptor B1 (SR-B1) (56), and the tight-junction proteins claudin 1 (CLDN1) (14) and occludin (OCLN) (54). The importance of E2 binding to the large extracellular loop of CD81 has been established in vitro (13,18,28,50,53), and interactions between E2 hypervariable region 1 (HVR1) and SR-B1

The liver-specific microRNA miR-122 is required for efficient hepatitis C virus (HCV) RNA replication both in cell culture and in vivo. In addition, nonhepatic cells have been rendered more efficient at supporting this stage of the HCV life cycle by miR-122 expression. This study investigated how miR-122 influences HCV replication in the miR-122-deficient HepG2 cell line. Expression of this microRNA in HepG2 cells permitted efficient HCV RNA replication and infectious virion production. When a missing HCV receptor is also expressed, these cells efficiently support viral entry and thus the entire HCV life cycle.Hepatitis C virus (HCV), a member of the family Flaviviridae, is associated with more than half of newly diagnosed hepatocellular carcinomas in the United States (1, 31) and is the leading cause of liver transplants worldwide (7). Historically, a lack of model systems to study HCV replication has slowed the progress of HCV research and the development of specific antivirals. Few HCV genomes replicate in culture, and only one efficiently produces infectious particles (23,39,42). Most model systems rely on the Huh-7 cell line and derivatives, such as the highly HCV-permissive Huh-7.5 subclone (6). The development of novel cell systems capable of supporting the entire HCV life cycle would allow broader examination of interactions between HCV and host cell biology and would provide essential tools for anti-HCV drug discovery.MicroRNAs (miRNAs) are small RNA molecules predicted to downregulate the expression of one-third of human genes by reducing mRNA stability and/or translation, depending on the degree of complementarity between the miRNA and the mRNA target site (2). miR-122, a liver-specific miRNA expressed at high levels in hepatocytes, is required to support HCV RNA replication (19). Two miR-122 target sites have been identified in the 5Ј untranslated region of the HCV genome, and mutation of these sites disrupts HCV RNA replication. Replication is restored by cotransfection with RNA duplexes that produce a mutant miR-122 with complementary changes (16)(17)(18)(19)26). How miR-122 enhances HCV replication is unclear. Although its binding appears to enhance HCV internal ribosome entry site (IRES)-directed translation, this effect is not great enough to account for the difference in RNA replication (16,17,40). Furthermore, the normal cellular function of miR-122 in regulating cholesterol and fatty acid biosynthesis is not required for this enhancement (18). Thus, miR-122 has been hypothesized to directly affect HCV RNA replication or stability (17,19,26). Although inhibiting miR-122 has been shown to be an effective therapy in chimpanzees infected with HCV (21), further development of such therapies will require a firmer understanding of how miR-122 promotes HCV replication.The majority of published studies on the role of miR-122 in the HCV life cycle have described research conducted with HCV-permissive Huh-7 cells or derivatives, which express endogenous miR-122 (19). Two studies explored the effect of m...

Hepatitis C virus (HCV) is a major cause of liver disease worldwide. A better understanding of its life cycle, including the process of host cell entry, is important for the development of HCV therapies and model systems. Based on the requirement for numerous host factors, including the two tight junction proteins claudin-1 (CLDN1) and occludin (OCLN), HCV cell entry has been proposed to be a multi-step process. The lack of OCLN-specific inhibitors has prevented a comprehensive analysis of this process. To study the role of OCLN in HCV cell entry, we created OCLN mutants whose HCV cell entry activities could be inhibited by antibodies. These mutants were expressed in polarized HepG2 cells engineered to support the complete HCV life cycle by CD81 and miR-122 expression and synchronized infection assays were performed to define the kinetics of HCV cell entry. During these studies, OCLN utilization differences between HCV isolates were observed, supporting a model that HCV directly interacts with OCLN. In HepG2 cells, both HCV cell entry and tight junction formation were impaired by OCLN silencing and restored by expression of antibody regulatable OCLN mutant. Synchronized infection assays showed that glycosaminoglycans and SR-BI mediated host cell binding, while CD81, CLDN1 and OCLN all acted sequentially at a post-binding stage prior to endosomal acidification. These results fit a model where the tight junction region is the last to be encountered by the virion prior to internalization.

Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. As HCV infects only human and chimpanzee cells, antiviral therapy and vaccine development have been hampered by the lack of a convenient small-animal model. In this study we further investigate how the species tropism of HCV is modulated at the level of cell entry. It has been previously determined that the tight junction protein occludin (OCLN) is essential for HCV host cell entry and that human OCLN is more efficient than the mouse ortholog at mediating HCV cell entry. To further investigate the relationship between OCLN sequence and HCV species tropism, we compared OCLN proteins from a range of species for their ability to mediate infection of naturally OCLNdeficient 786-O cells with lentiviral pseudoparticles bearing the HCV glycoproteins. While primate sequences function equivalently to human OCLN, canine, hamster, and rat OCLN had intermediate activities, and guinea pig OCLN was completely nonfunctional. Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein. Furthermore, this critical region of EC2 is flanked by two conserved cysteine residues that are essential for HCV cell entry, suggesting that a subdomain of EC2 may be defined by a disulfide bond.Hepatitis C virus (HCV), a member of the family Flaviviridae, is the causative agent of classically defined non-A, non-B hepatitis and is highly prevalent, with approximately 3% of the worldwide population infected (48). HCV infection often results in a chronic, life-long infection that can have severe health consequences, including hepatitis, cirrhosis, hepatocellular carcinoma, and liver failure. There is no HCV vaccine available, and the currently employed interferon-based treatment is inadequate as it has severe side effects and is effective only in half of the major genotype-infected individuals (22,32). Specific anti-HCV inhibitors targeting the viral proteases and polymerase are currently being developed and will likely improve therapeutic options substantially. Undoubtedly, however, the emergence of viral resistance to such inhibitors will be a problem facing future HCV treatment options. As such, developing a spectrum of inhibitors targeting diverse steps in the virus life cycle, including HCV cell entry, is a priority for HCV research. Such inhibitors may be particularly useful following liver transplantation. Although HCV is the leading cause of liver transplants worldwide (10), the usefulness of such procedures is limited by subsequent universal graft reinfection and often accelerated disease progression (21). Even transiently inhibiting graft reinfection with HCV cell entry inhibitors could greatly improve the effectiveness of this procedure. Therefore, a greater understanding of HCV cell entry is required for the ...