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

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

Identification of a Human Respiratory Syncytial Virus Cell Entry Inhibitor by Using a Novel Lentiviral Pseudotype System.

Lentiviral budding is governed by group-specific antigens (Gag proteins) and proceeds in the absence of cognate viral envelope proteins, which has been exploited to create pseudotypes incorporating envelope proteins from nonlentiviral families. Here, we report the generation of infectious lentiviral pseudoparticles incorporating human respiratory syncytial virus (hRSV) F protein alone (hRSV-Fpp) or carrying SH, G, and F proteins (hRSV-SH/G/Fpp). These particles recapitulate key infection steps of authentic hRSV particles, including utilization of glycosaminoglycans and low-pH-independent cell entry. Moreover, hRSV pseudoparticles (hRSVpp) can faithfully reproduce phenotypic resistance to a small-molecule fusion inhibitor in clinical development (BMS-433771) and a licensed therapeutic F protein-targeting antibody (palivizumab). Inoculation of several human cell lines from lung and liver revealed more than 30-fold differences in susceptibility to hRSVpp infection, suggesting differential expression of hRSV entry cofactors and/or restriction factors between these cell types. Moreover, we observed cell-type-dependent functional differences between hRSVpp carrying solely F protein or SH, G, and F proteins with regard to utilization of glycosaminoglycans. Using hRSVpp, we identified penta-O-galloyl-β-d-glucose (PGG) as a novel hRSV cell entry inhibitor. Moreover, we show that PGG also inhibits cell entry of hRSVpp carrying F proteins resistant to BMS-433771 or palivizumab. This work sheds new light on the mechanisms of hRSV cell entry, including possible strategies for antiviral intervention. Moreover, hRSVpp should prove valuable to dissect hRSV envelope protein functions, including the interaction with cell entry factors. Importance: Lentiviral pseudotypes are highly useful to specifically dissect the functions of viral and host factors in cell entry, which have been exploited for numerous viruses. Here, we successfully created hRSVpp and show that they faithfully recapitulate key characteristics of parental hRSV cell entry. Importantly, hRSVpp accurately mirror hRSV resistance to small-molecule fusion inhibitors and clinically approved therapeutic antibodies. Moreover, we observed highly different susceptibilities of cell lines to hRSVpp infection and also differences between hRSVpp types (with F protein alone or with SH, G, and F proteins) in regard to cell entry. This indicates differential expression of host factors determining hRSV cell entry between these cell lines and highlights the fact that the hRSVpp system is useful to explore the functional properties of hRSV envelope protein combinations. Therefore, this system will be highly useful to study hRSV cell entry and host factor usage and to explore antiviral strategies targeting hRSV cell entry

Magnesium Complexes of Ladanein: A Beneficial Strategy for Stabilizing Polyphenolic Antivirals

Ladanein (noted FOMe) is a potent antiviral flavone that was shown to be active on a broad spectrum of enveloped viruses. This 5,6,7-trihydroxylated flavone has, however, pharmacokinetic properties and a half-life time that need to be improved for possible therapeutic applications. We herein took advantage of the complexation properties of ladanein (Fe(III)) to evaluate its ability to bind Mg(II) (biologically relevant and redox inert ion) precursors prepared beforehand from various carboxylic acids. The 5,6,7-trihydroxylated pattern of ladanein and the ligands borne by the Mg(II) atom of the precursors were found to be essential for firm Mg(II) binding. In particular, a ternary Mg(II) complex of ladanein and pidolate (noted FOMe.MgPid) was isolated and considered for its pharmacokinetic and virucidal (Hepatitis C Virus - HCV) properties. Mg(II) complexation significantly improved the physico-chemical (solubility) and the pharmacokinetic properties (clearance, plasmatic concentration) of the flavone FOMe, while not altering its anti-HCV capacity.Université de Strasbour

Efficient virus assembly, but not infectivity, determines the magnitude of hepatitis C virus-induced interferon alpha responses of plasmacytoid dendritic cells.

Worldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses 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

High affinity peptide inhibitors of the hepatitis C virus NS3-4A protease refractory to common resistant mutants.

Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel "tyrosine" finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K(i) of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease

Thiamyxins: Structure and Biosynthesis of Myxobacterial RNA-Virus-Inhibitors

During our search for novel myxobacterial natural products, we discovered the Thiamyxins: thiazole- and thiazoline-rich non-ribosomal peptide-polyketide hybrids with potent antiviral activity. We isolated two cyclized and two open-chain congeners of this unprecedented natural product family, whereof the non-cyclized Thiamyxin D was found to be fused to a glycerol unit attached to the C-terminal carboxyl function. Alongside their structure elucidation and absolute stereochemistry, we present the biosynthetic origin of the Thiamyxins supported by a concise biosynthesis model based on biosynthetic gene cluster analysis and feeding experiments with isotope labelled precursors. We report an unprecedented incorporation of a 2-(hydroxymethyl)-4-methylpent-3-enoic acid moiety originating from the involved polyketide synthase featuring a rare GCN5-related N-acetyltransferase-like decarboxylase domain. The Thiamyxins showed potent inhibition of different RNA-viruses as analysed in cell culture models of corona, zika and dengue virus infection. Their potency up to a half maximal inhibitory concentration of 560 nM combined with milder cytotoxic effects on human cell lines indicate a potential for further development of the Thiamyxins as broad-spectrum antivirals targeting RNA-viruses