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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.

Recent emergence of direct-acting antivirals (DAAs) targeting hepatitis C virus (HCV) proteins has considerably enhanced the success of antiviral therapy. However, the appearance of DAA-resistant-associated variants is a cause of treatment failure, and the high cost of DAAs renders the therapy not accessible in countries with inadequate medical infrastructures. Therefore, the search for new inhibitors with a lower cost of production should be pursued. In this context, the crude extract of Juncus maritimus Lam. was shown to exhibit high antiviral activity against HCV in cell culture. Bio-guided fractionation allowed the isolation and identification of the active compound, dehydrojuncusol. A time-of-addition assay showed that dehydrojuncusol significantly inhibited HCV infection when added after virus inoculation of HCV genotype 2a (50% effective concentration [EC 50 ] = 1.35 µM). This antiviral activity was confirmed with an HCV subgenomic replicon, and no effect on HCV pseudoparticle entry was observed. Antiviral activity of dehydrojuncusol was also demonstrated in primary human hepatocytes. No in vitro toxicity was observed at active concentrations. Dehydrojuncusol is also efficient on HCV genotype 3a and can be used in combination with sofosbuvir. Interestingly, dehydrojuncusol was able to inhibit RNA replication of two frequent daclatasvir-resistant mutants (L31M or Y93H in NS5A). Finally, mutants resistant to dehydrojuncusol were obtained and showed that the HCV NS5A protein is the target of the molecule. In conclusion, dehydrojuncusol, a natural compound extracted from J. maritimus , inhibits infection of different HCV genotypes by targeting the NS5A protein and is active against resistant HCV variants frequently found in patients with treatment failure. IMPORTANCE Tens of millions of people are infected with hepatitis C virus (HCV) worldwide. Recently marketed direct-acting antivirals (DAAs) targeting HCV proteins have enhanced the efficacy of treatment. However, due to its high cost, this new therapy is not accessible to the vast majority of infected patients. Furthermore, treatment failures have also been reported due to the appearance of viral resistance. Here, we report on the identification of a new HCV inhibitor, dehydrojuncusol, that targets HCV NS5A and is able to inhibit RNA replication of replicons harboring resistance mutations to anti-NS5A DAAs used in current therapy. Dehydrojuncusol is a natural compound isolated from Juncus maritimus , a halophilic plant species that is very common in coastlines worldwide. This molecule might serve as a lead for the development of a new therapy that is more accessible to hepatitis C patients in the future., (Copyright © 2019 American Society for Microbiology.)

Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells. The relevance of cell polarity in the HCV life cycle has been addressed only in distantly related models and remains poorly understood. Although polarized epithelial cells have a rather simple morphology with a basolateral and an apical domain, hepatocytes exhibit complex polarization structures. However, it has been reported that some selected polarized HepG2 cell clones can exhibit a honeycomb pattern of distribution of the tight-junction proteins typical of columnar polarized epithelia, which can be used as a simple model to study the role of cell polarization in viral infection of hepatocytes. To obtain similar clones, HepG2 cells expressing CD81 (HepG2-CD81) were used, and clones were isolated by limiting dilutions. Two clones exhibiting a simple columnar polarization capacity when grown on a semipermeable support were isolated and characterized. To test the polarity of HCV entry and release, our polarized HepG2-CD81 clones were infected with cell culture-derived HCV. Our data indicate that HCV binds equally to both sides of the cells, but productive infection occurs mainly when the virus is added at the basolateral domain. Furthermore, we also observed that HCV virions are released from the basolateral domain of the cells. Finally, when polarized cells were treated with oleic acid and U0126, a MEK inhibitor, to promote lipoprotein secretion, a higher proportion of infectious viral particles of lower density were secreted. This cell culture system provides an excellent model to investigate the influence of cell polarization on the HCV life cycle. IMPORTANCE Hepatitis C is a major health burden, with approximately 170 million persons infected worldwide. Hepatitis C virus (HCV) primarily infects hepatocytes, which are highly polarized cells with a complex organization. The relevance of cell polarity in the HCV life cycle has been addressed in distantly related models and remains unclear. Hepatocyte organization is complex, with multiple apical and basolateral surfaces. A simple culture model of HepG2 cells expressing CD81 that are able to polarize with unique apical and basolateral domains was developed to study HCV infection. With this model, we demonstrated that HCV enters and exits hepatocytes by the basolateral domain. Furthermore, lower-density viral particles were produced under conditions that promote lipoprotein secretion. This cell culture system provides a useful model to study the influence of cell polarization on HCV infection., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Aminoquinolines and piperazines, linked or not, have been used successfully to treat malaria, and some molecules of this family also exhibit antiviral properties. Here we tested several derivatives of 4-aminoquinolines and piperazines for their activity against hepatitis C virus (HCV). We screened 11 molecules from three different families of compounds, and we identified anti-HCV activity in cell culture for six of them. Of these, we selected a compound (B5) that is currently ending clinical phase I evaluation for neurodegenerative diseases. In hepatoma cells, B5 inhibited HCV infection in a pangenotypic and dose-dependent manner, and its antiviral activity was confirmed in primary hepatocytes. B5 also inhibited infection by pseudoparticles expressing HCV envelope glycoproteins E1 and E2, and we demonstrated that it affects a postattachment stage of the entry step. Virus with resistance to B5 was selected by sequential passage in the presence of the drug, and reverse genetics experiments indicated that resistance was conferred mainly by a single mutation in the putative fusion peptide of E1 envelope glycoprotein (F291I). Furthermore, analyses of the effects of other closely related compounds on the B5-resistant mutant suggest that B5 shares a mode of action with other 4-aminoquinoline-based molecules. Finally, mice with humanized liver that were treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle., Importance: In the last 4 years, HCV therapy has been profoundly improved with the approval of direct-acting antivirals in clinical practice. Nevertheless, the high costs of these drugs limit access to therapy in most countries. The present study reports the identification and characterization of a compound (B5) that inhibits HCV propagation in cell culture and is currently ending clinical phase I evaluation for neurodegenerative diseases. This molecule inhibits the HCV life cycle by blocking virus entry. Interestingly, after selection of drug-resistant virus, a resistance mutation in the putative fusion peptide of E1 envelope glycoprotein was identified, indicating that B5 could be used to further investigate the fusion mechanism. Furthermore, mice with humanized liver treated with B5 showed a delay in the kinetics of the viral infection. In conclusion, B5 is a novel interesting anti-HCV molecule that could be used to decipher the early steps of the HCV life cycle., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Despite the validation of direct-acting antivirals for hepatitis C treatment, the discovery of new compounds with different modes of action may still be of importance for the treatment of special patient populations. We recently identified a natural molecule, epigallocatechin-3-gallate (EGCG), as an inhibitor of hepatitis C virus (HCV) targeting the viral particle. The aim of this work was to discover new natural compounds with higher anti-HCV activity than that of EGCG and determine their mode of action. Eight natural molecules with structure similarity to EGCG were selected. HCV JFH1 in cell culture and HCV pseudoparticle systems were used to determine the antiviral activity and mechanism of action of the compounds. We identified delphinidin, a polyphenol belonging to the anthocyanidin family, as a new inhibitor of HCV entry. Delphinidin inhibits HCV entry in a pangenotypic manner by acting directly on the viral particle and impairing its attachment to the cell surface. Importantly, it is also active against HCV in primary human hepatocytes, with no apparent cytotoxicity and in combination with interferon and boceprevir in cell culture. Different approaches showed that neither aggregation nor destruction of the particle occurred. Cryo-transmission electron microscopy observations of HCV pseudoparticles treated with delphinidin or EGCG showed a bulge on particles that was not observed under control conditions. In conclusion, EGCG and delphinidin inhibit HCV entry by a new mechanism, i.e., alteration of the viral particle structure that impairs its attachment to the cell surface., Importance: In this article, we identify a new inhibitor of hepatitis C virus (HCV) infection, delphinidin, that prevents HCV entry. This natural compound, a plant pigment responsible for the blue-purple color of flowers and berries, belongs to the flavonoid family, like the catechin EGCG, the major component present in green tea extract, which is also an inhibitor of HCV entry. We studied the mode of action of these two compounds against HCV and demonstrated that they both act directly on the virus, inducing a bulging of the viral envelope. This deformation might be responsible for the observed inhibition of virus attachment to the cell surface. The discovery of such HCV inhibitors with an unusual mode of action is important to better characterize the mechanism of HCV entry into hepatocytes and to help develop a new class of HCV entry inhibitors., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Hepatitis C virus (HCV) entry involves binding to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction in the context of a viral infection. Patient sera and monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pulldown assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with the virion-heparin interaction, strongly suggesting that at the virion surface, HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pulldown experiments, and inhibition experiments with anti-apolipoprotein E antibodies indicated that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of the HS structural determinants required for HCV infection by silencing of the enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation is required for HCV infection and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures., Importance: Hepatitis C is a global health problem. Hepatitis C virus (HCV) infects approximately 130 million individuals worldwide, with the majority of cases remaining undiagnosed and untreated. In most infected individuals, the virus evades the immune system and establishes a chronic infection. As a consequence, hepatitis C is the leading cause of cirrhosis, end-stage liver disease, hepatocellular carcinoma, and liver transplantation. Virus infection is initiated by entry of the virus into the host cell. In this study, we provide new insights into the viral and cellular determinants involved in the first step of HCV entry, the binding of the virus to host cells. We show that apolipoprotein E is likely responsible for virus binding to heparan sulfate and that N- and 6-O-sulfation of the heparan sulfate proteoglycans is required for HCV infection. In addition, the minimal HS length unit required for HCV infection is a decasaccharide., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: In spite of the high variability of its sequence, hepatitis C virus (HCV) envelope glycoprotein E2 contains several conserved regions. In this study, we explored the structural and functional features of the highly conserved E2 segment from amino acid (aa) 502 to 520, which had been proposed as a fusion peptide and shown to strongly overlap a potential conserved neutralizing epitope. For this purpose, we used reverse genetics to introduce point mutations within this region, and we characterized the phenotypes of these mutants in the light of the recently published structure of E2. The functional analyses showed that their phenotypes are in agreement with the positions of the corresponding residues in the E2 crystal structure. In contrast, our data ruled out the involvement of this region in membrane fusion, and they indicate that alternative conformations would be necessary to expose the potential neutralizing epitope present in this segment. Of particular interest, we identified three specific mutations (Y507L, V514A, and V515A) located within this neutralizing epitope which only mildly reduced infectivity and showed no assembly defect. These mutations modulated HCV dependence on the viral receptor SRB1, and/or they also modulated virion sensitivity to neutralizing antibodies. Importantly, their characterization also showed that amino acids Y507, V514, and V515 contribute to E2 interaction with HCV receptor CD81. In conclusion, our data show that the highly conserved E2 segment from aa 502 to 520 plays a key role in cell entry by influencing the association of the viral particle with coreceptors and neutralizing antibodies., Importance: Hepatitis C virus (HCV) envelope proteins E1 and E2 exhibit sequence variability. However, some segments of the envelope proteins are highly conserved, suggesting that these sequences play a key role at some steps of the HCV life cycle. In this work, we characterized the function and structure of a highly conserved E2 region that is targeted by neutralizing antibodies and had been proposed as a fusion peptide. Our data ruled out the involvement of this region in membrane fusion but allowed for the identification of new residues modulating the interaction of the virus with entry factors and its sensitivity to neutralizing antibodies. Moreover, structural data suggest that alternative conformations could exist for E2, which would explain the presence of a partially masked neutralizing epitope in this segment in the currently available E2 structure. Overall, our findings highlight the importance of conserved regions in the sequences of HCV envelope proteins., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In Saccharomyces cerevisiae the FUR4-encoded uracil permease catalyzes the first step of the pyrimidine salvage pathway. The availability of uracil has a negative regulatory effect upon its own transport. Uracil causes a decrease in the level of uracil permease, partly by decreasing the FUR4 mRNA level in a promoter-independent fashion, probably by increasing its instability. Uracil entry also triggers more rapid degradation of the existing permease by promoting high efficiency of ubiquitination of the permease that signals its internalization. A direct binding of intracellular uracil to the permease is possibly involved in this feedback regulation, as the behavior of the permease is similar in mutant cells unable to convert intracellular uracil into UMP. We used cells impaired in the ubiquitination step to show that the addition of uracil produces rapid inhibition of uracil transport. This may be the first response prior to the removal of the permease from the plasma membrane. Similar down-regulation of uracil uptake, involving several processes, was observed under adverse conditions mainly corresponding to a decrease in the cellular content of ribosomes. These results suggest that uracil of exogenous or catabolic origin down-regulates the cognate permease to prevent buildup of excess intracellular uracil-derived nucleotides.