Sibylle Haid

Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors.

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.

MAP-Kinase Regulated Cytosolic Phospholipase A2 Activity Is Essential for Production of Infectious Hepatitis C Virus Particles

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.

Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles

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.

Apolipoprotein E Codetermines Tissue Tropism of Hepatitis C Virus and Is Crucial for Viral Cell-to-Cell Transmission by Contributing to a Postenvelopment Step of Assembly

ABSTRACT Hepatitis 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 (HCVTCP) 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.

A Plant-Derived Flavonoid Inhibits Entry of All HCV Genotypes Into Human Hepatocytes

BACKGROUND & AIMS Interferon-based therapies for hepatitis C virus (HCV) infection are limited by side effects and incomplete response rates, particularly among transplant recipients. We screened a library of plant-derived small molecules to identify HCV inhibitors with novel mechanisms. METHODS We isolated phenolic compounds from Marrubium peregrinum L (Lamiaceae). Replication of HCV RNA, virus production, and cell entry were monitored using replicons and infectious HCV. Inhibition of HCV was measured in hepatoma cells and primary human hepatocytes using luciferase reporter gene assays, core enzyme-linked immunosorbent assays, or infectivity titration. We tested the bioavailability of the compound in mice. RESULTS We identified a flavonoid, ladanein (BJ486K), with unreported antiviral activity and established its oral bioavailability in mice. Natural and synthetic BJ486K inhibited a post-attachment entry step, but not RNA replication or assembly; its inhibitory concentration 50% was 2.5 μm. BJ486K was effective against all major HCV genotypes, including a variant that is resistant to an entry inhibitor; it prevented infection of primary human hepatocytes. Combined administration of BJ486K and cyclosporine A had a synergistic effect in inhibition of HCV infection. CONCLUSIONS BJ486K has oral bioavailability and interferes with entry of HCV into cultured human hepatocytes. It synergizes with cyclosporine A to inhibit HCV infection. Its inhibitory effects are independent of HCV genotype, including a variant that is resistant to an entry inhibitor against scavenger receptor class B type I. Flavonoid derivatives therefore might be developed as components of combination therapies because they are potent, broadly active inhibitors of HCV entry that could prevent graft reinfection after liver transplantation.

Mouse-Specific Residues of Claudin-1 Limit Hepatitis C Virus Genotype 2a Infection in a Human Hepatocyte Cell Line

ABSTRACT Recently, claudin-1 (CLDN1) was identified as a host protein essential for hepatitis C virus (HCV) infection. To evaluate CLDN1 function during virus entry, we searched for hepatocyte cell lines permissive for HCV RNA replication but with limiting endogenous CLDN1 expression, thus permitting receptor complementation assays. These criteria were met by the human hepatoblastoma cell line HuH6, which (i) displays low endogenous CLDN1 levels, (ii) efficiently replicates HCV RNA, and (iii) produces HCV particles with properties similar to those of particles generated in Huh-7.5 cells. Importantly, naïve cells are resistant to HCV genotype 2a infection unless CLDN1 is expressed. Interestingly, complementation of HCV entry by human, rat, or hamster CLDN1 was highly efficient, while mouse CLDN1 (mCLDN1) supported HCV genotype 2a infection with only moderate efficiency. These differences were observed irrespective of whether cells were infected with HCV pseudoparticles (HCVpp) or cell culture-derived HCV (HCVcc). Comparatively low entry function of mCLDN1 was observed in HuH6 but not 293T cells, suggesting that species-specific usage of CLDN1 is cell type dependent. Moreover, it was linked to three mouse-specific residues in the second extracellular loop (L152, I155) and the fourth transmembrane helix (V180) of the protein. These determinants could modulate the exposure or affinity of a putative viral binding site on CLDN1 or prevent optimal interaction of CLDN1 with other human cofactors, thus precluding highly efficient infection. HuH6 cells represent a valuable model for analysis of the complete HCV replication cycle in vitro and in particular for analysis of CLDN1 function in HCV cell entry.

Isolate‐dependent use of claudins for cell entry by hepatitis C virus

Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB‐1), occludin (OCLN), and claudin‐1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh‐7.5) or CLDN6 (HuH6). Huh‐7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6‐dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1‐specific neutralizing antibodies (Abs) fully abrogated infection of Huh‐7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti‐CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1‐specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB‐1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. Conclusion: These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1‐specific therapeutic strategies. (Hepatology 2014;58:24–34)

Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1.

To explore mechanisms of hepatitis C viral (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 a cost‐effective component of HCV combination therapies. (Hepatology 2016;63:49–62)

Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

Role of Hypervariable Region 1 for the Interplay of Hepatitis C Virus with Entry Factors and Lipoproteins

ABSTRACT 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. IMPORTANCE HCV cell entry is SR-BI dependent irrespective of the presence or absence of HVR1. Moreover, this domain modulates the properties of ApoE on the surface of virus particles. These findings have implications for the development of SR-BI-targeting antivirals. Furthermore, these findings highlight separable functions of SR-BI during HCV cell entry and reveal a novel role of HVR1 for the properties of virus-associated lipoproteins.