Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo.

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 µM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

Hepatitis C virus infection propagates through interactions between Syndecan-1 and CD81 and impacts the hepatocyte glycocalyx.

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.

Arbidol as a broad-spectrum antiviral: an update.

Arbidol (ARB) is a Russian-made small indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It also demonstrates inhibitory activity against other viruses, enveloped or not, responsible for emerging or globally prevalent infectious diseases such as hepatitis B and C, gastroenteritis, hemorrhagic fevers or encephalitis. In this review, we will explore the possibility and pertinence of ARB as a broad-spectrum antiviral, after a careful examination of its physico-chemical properties, pharmacokinetics, toxicity, and molecular mechanisms of action. Recent studies suggest that ARB's dual interactions with membranes and aromatic amino acids in proteins may be central to its broad-spectrum antiviral activity. This could impact on the virus itself, and/or on cellular functions or critical steps in virus-cell interactions, thereby positioning ARB as both a direct-acting antiviral (DAA) and a host-targeting agent (HTA). In the context of recent studies in animals and humans, we will discuss the prospective clinical use of ARB in various viral infections.

Arbidol inhibits viral entry by interfering with clathrin-dependent trafficking.

Arbidol (ARB) is a broad-spectrum antiviral displaying activity against a number of enveloped and non-enveloped viruses. It was described as a viral entry inhibitor and shown to interact at the molecular level with lipid membranes and viral fusion glycoproteins to impede viral entry and fusion. However its mechanism of action at the cellular level remains unknown. Here, by using live-cell confocal imaging and the hepatitis C virus as a model virus, we show that ARB affects clathrin-mediated endocytosis by impeding dynamin-2-induced membrane scission. Moreover it induces the intracellular accumulation of clathrin-coated structures where viral particles are trapped. Collectively, our results shed light on the mechanistic aspects of ARB antiviral activity and suggest that ARB could prevent cell infection by viruses that enter through clathrin-mediated endocytosis.

Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin-dependent trafficking.

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti-HCV action of silymarin-derived compounds at the molecular level. By using live-cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV-infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin-coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin-coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well-known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin-mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad-spectrum antiviral therapy.

Lipids: a key for hepatitis C virus entry and a potential target for antiviral strategies.

Viruses have evolved to complex relationship with their host cells. Many viruses modulate the lipid composition, lipid synthesis and signaling of their host cell. Lipids are also an essential part of the life cycle of the hepatitis C virus (HCV). HCV is a major human pathogen, persistently infecting 170 million people worldwide, with no currently effective treatment available for all patients. HCV appears to make use of the host lipid metabolism and one common feature of chronic hepatitis C is the steatosis, characterized by excessive accumulation of triglycerides and lipid content in the liver. Thus, HCV lifecycle appears to be closely connected to host cell lipid metabolism, from cell entry, through viral RNA replication to viral particle production and formation/assembly. HCV particles have a unique lipid composition, certainly distinct from other viruses. In the blood of chronically-infected patients, viral particles are bound to serum lipoproteins and are thus called lipo-viro-particles. The density of these circulating viral particles is heterogeneous. Specific infectivity and fusion of low density particles are greater than those of high density particles. Lipids and association to lipoproteins therefore play a key role in HCV life cycle. The purpose of this review is to make a state of the art on recent findings on the contribution of lipids in cell entry and membrane fusion of HCV. The influence of lipids as chemically-defined entities will be analyzed, as well as the role played by cholesterol transporters and lipoprotein receptors in HCV entry and fusion. Since viral entry would constitute a key target for antiviral strategies, inhibitor molecules interacting with viral and/or cellular membranes or interfering with the function of lipid metabolism regulators of HCV entry could offer strong antiviral potential. This will be lastly discussed in this review.