Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.

cHepatitis C virus (HCV) predominantly infects human hepatocytes, although extrahepatic virus reservoirs are being discussed. Infection of cells is initiated via cell-free and direct cell-to-cell transmission routes. Cell type-specific determinants of HCV entry and RNA replication have been reported. Moreover, several host factors required for synthesis and secretion of lipoproteins from liver cells, in part expressed in tissue-specific fashion, have been implicated in HCV assembly. However, the minimal cell type-specific requirements for HCV assembly have remained elusive. Here we report that production of HCV trans-complemented particles (HCV TCP ) from nonliver cells depends on ectopic expression of apolipoprotein E (ApoE). For efficient virus production by full-length HCV genomes, microRNA 122 (miR-122)-mediated enhancement of RNA replication is additionally required. Typical properties of cell culture-grown HCV (HCVcc) particles from ApoE-expressing nonliver cells are comparable to those of virions derived from human hepatoma cells, although specific infectivity of virions is modestly reduced. Thus, apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTTP), and apolipoprotein C1 (ApoC1), previously implicated in HCV assembly, are dispensable for production of infectious HCV. In the absence of ApoE, release of core protein from infected cells is reduced, and production of extracellular as well as intracellular infectivity is ablated. Since envelopment of capsids was not impaired, we conclude that ApoE acts after capsid envelopment but prior to secretion of infectious HCV. Remarkably, the lack of ApoE also abrogated direct HCV cell-to-cell transmission. These findings highlight ApoE as a host factor codetermining HCV tissue tropism due to its involvement in a late assembly step and viral cell-to-cell transmission. C urrently, around 160 million people are chronically infected with hepatitis C virus (HCV) worldwide (1). A prophylactic vaccine is not available, but direct-acting antivirals (DAA) have recently been approved for treatment (2). However, the novel triple therapy including pegylated alpha interferon (PEG-IFN-␣), ribavirin, and one of two available protease inhibitors is associated with side effects and is not licensed for all viral genotypes. A detailed understanding of the viral life cycle and the roles of specific viral and host factors may reveal novel targets for antiviral therapy and thus help to improve existing therapeutic options.Chronic HCV infection is a leading cause of liver disease, including hepatitis, liver cirrhosis, and hepatocellular carcinoma (3). It is also associated with numerous extrahepatic manifestations, such as cryoglobulenimia and neuronal disorders (reviewed in reference 4). While hepatocytes are thought to be the primary site of HCV replication, a number of studies have highlighted possible extrahepatic sites of replication, including peripheral blood mononuclear cells and cells of neuronal origin (reviewed in references 5 and 6). These observations sugge...

Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny.

To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies.

Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can be triggered in a receptor-independent but pH-dependent manner and that fusion of the HCV particles with liposomes is dependent on the viral dose and on the lipid composition of the target membranes. In addition CBH-5, an HCV E2-specific antibody, inhibited fusion in a dose-dependent manner. Interestingly, point mutations in E2, known to abrogate HCV glycoprotein-mediated fusion in a cell-based assay, altered or even abolished fusion in the liposome-based assay. When assaying the fusion properties of HCV particles with different buoyant density, we noted higher fusogenicity of particles with lower density. This could be attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-floatation of factors enhancing fusion activity in trans. Taken together, these data show the important role of lipids of both the viral and target membranes in HCV-mediated fusion, point to a crucial role played by the E2 glycoprotein in the process of HCV fusion, and reveal an important behavior of HCV of different densities with regard to fusion. Hepatitis C virus (HCV)4 is an important public health concern worldwide as it is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped virus that belongs to the Hepacivirus genus of the Flaviviridae family (1). Based on sequence comparison, patient isolates are classified into seven genotypes, differing in their nucleotide sequence by 30 -35% (2-5). The two viral surface proteins, E1 (residues 192-383) and E2 (residues 384 -746), are processed by signal peptidases of the endoplasmic reticulum from a 3,000-amino acid-long polyprotein encoded by the HCV genome (reviewed in Ref.2). The E1 (ϳ31 kDa) and E2 (ϳ70 kDa) proteins are glycosylated in their large amino-terminal ectodomains (6) and are anchored in the viral membrane by their carboxyl-terminal transmembrane domains. E1 and E2 form a heterodimer stabilized by noncovalent interactions. This oligomer is thought to be present at the surface of HCV particles (7) and to be involved in viral entry. Carboxyl-terminally truncated soluble E2 protein is known to specifically bind to crucial HCV entry factors like glycosaminoglycans, the tetraspanin CD81, and the scavenger receptor BI (8 -12). Thus, virus-associated E2 is likely directly involved in interactions important for virus attachment and productive infection (reviewed in Refs. 13,14).Both HCV envelope glycoproteins are the targets for virusneutralizing antibodies (7,(15)...

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Biomolecular condensates have emerged as an important subcellular organizing principle 1 . Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm 2,3 . IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation 4,5 . Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers 6 .RSV is a major cause of respiratory illness in young children, the older people and individuals who are immunocompromised worldwide 7,8 . Currently, multiple targets are pursued for the development of a safe and effective therapy to treat RSV infections 9 .In infected cells, RSV induces the formation of cytoplasmic IBs, in which nucleoprotein (N), phosphoprotein (P), polymerase L, the transcription factor M2-1 and viral genomic RNA are concentrated. We recently demonstrated that IBs are 'viral factories' in which viral RNA synthesis occurs 3 . The morphology of IBs suggests that they are condensates formed by liquid-liquid phase separation (LLPS). A recent study showed that N and P were sufficient to drive the formation of pseudo-IB condensates through LLPS in vitro, both in cells and in biochemical assays 10 . However, these N-P pseudo-IB condensates are not functional, as they do not shelter RNA synthesis and do not reflect the complexity of IBs in virus-infected cells, which have multiple compartments. Strikingly similar in size and phase organization to the nucleolus condensate 11 , RSV IBs are multiphasic and contain a sub-compartment called the IB-associated granule (IBAG), which is composed of newly synthesized viral mRNA and M2-1 3,12 . Condensates have emerged as an important subcellular organizing principle 1 . An important question in anti-viral drug developmentand medicinal chemistry more generally-is whether these condensates are druggable. In principle, a drug that dissolved or hardened would prevent viral replication. Neither mechanism has yet been reported. Chemical analogues without hedgehog antagonismWe previously identified the hedgehog (HH) pathway antagonist cyclopamine (CPM) as a potent inhibitor of RSV replication 13 . Inhibition of Sonic hedgehog (SHH) signalling is an unwanted feature of CPM as an RSV inhibitor. On the basis of the binding model of the Smoothe...

Hepatitis C virus (HCV) particles associate with lipoproteins and infect cells by using at least four cell entry factors. These factors include scavenger receptor class B type I (SR-BI), CD81, claudin 1 (CLDN1), and occludin (OCLN). Little is known about specific functions of individual host factors during HCV cell entry and viral domains that mediate interactions with these factors. Hypervariable region 1 (HVR1) within viral envelope protein 2 (E2) is involved in the usage of SR-BI and conceals the viral CD81 binding site. Moreover, deletion of this domain alters the density of virions. We compared lipoprotein interaction, surface attachment, receptor usage, and cell entry between wild-type HCV and a viral mutant lacking this domain. Deletion of HVR1 did not affect CD81, CLDN1, and OCLN usage. However, unlike wild-type HCV, HVR1-deleted viruses were not neutralized by antibodies and small molecules targeting SR-BI. Nevertheless, modulation of SR-BI cell surface expression altered the infection efficiencies of both viruses to similar levels. Analysis of affinity-purified virions revealed comparable levels of apolipoprotein E (ApoE) incorporation into viruses with or without HVR1. However, ApoE incorporated into these viruses was differentially recognized by ApoE-specific antibodies. Thus, SR-BI has at least two functions during cell entry. One of them can be neutralized by SR-BI-targeting molecules, and it is critical only for wild-type HCV. The other one is important for both viruses but apparently is not inactivated by the SR-BI binding antibodies and small molecules evaluated here. In addition, HVR1 modulates the conformation and/or epitope exposure of virus particle-associated ApoE.