Menashe Elazar

Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (Kd) of ∼3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4Bs RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

Amphipathic Helix-Dependent Localization of NS5A Mediates Hepatitis C Virus RNA Replication

ABSTRACT We identified an N-terminal amphipathic helix (AH) in one of hepatitis C virus (HCV)’s nonstructural proteins, NS5A. This AH is necessary and sufficient for membrane localization and is conserved across isolates. Genetically disrupting the AH impairs HCV replication. Moreover, an AH peptide-mimic inhibits the membrane association of NS5A in a dose-dependent manner. These results have exciting implications for the HCV life cycle and novel antiviral strategies.

An N-Terminal Amphipathic Helix in Hepatitis C Virus (HCV) NS4B Mediates Membrane Association, Correct Localization of Replication Complex Proteins, and HCV RNA Replication

ABSTRACT Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the viruss nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AHs ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4Bs N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.

A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

ABSTRACT Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the viruss nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies.

The hepatitis C virus NS5A inhibitor (BMS-790052) alters the subcellular localization of the NS5A non-structural viral protein

The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs.

A Rab-GAP TBC Domain Protein Binds Hepatitis C Virus NS5A and Mediates Viral Replication

ABSTRACT Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the worlds population.

The Anti-Hepatitis C Agent Nitazoxanide Induces Phosphorylation of Eukaryotic Initiation Factor 2α Via Protein Kinase Activated by Double-Stranded RNA Activation

BACKGROUND & AIMS New therapies are needed to treat patients infected with hepatitis C virus (HCV), a major worldwide cause of chronic liver disease. Nitazoxanide (NTZ), originally used to treat cryptosporidiosis infection, recently was shown to have unexpected antiviral activity in the HCV replicon system and in chronically infected patients. A pilot clinical study suggested that NTZ can augment the antiviral effect of interferon (IFN), although the molecular basis for its effect was unknown. METHODS We analyzed the effects of NTZ on the regulation of eukaryotic initiation factor-2alpha (eIF2alpha) and its IFN-induced kinase, protein kinase activated by double-stranded RNA (PKR), in cells that support HCV RNA replication and in vitro biochemical assays. RESULTS NTZ increased eIF2alpha phosphorylation, a modification known to mediate host cell antiviral defenses. The addition of IFN to cell cultures increased NTZ-induced eIF2alpha phosphorylation. NTZ also increased PKR phosphorylation. In vitro, NTZ promoted PKR autophosphorylation, a key step in activating PKRs kinase activity for eIF2alpha. Finally, NTZ-induced eIF2alpha phosphorylation was reduced in the presence of specific inhibitors of PKR autophosphorylation. CONCLUSIONS An important mechanism of NTZs action involves activation of PKR, a key kinase that regulates the cells innate antiviral response. These observations could explain the clinical antiviral effect of NTZ. NTZ might represent a new class of small molecules capable of potentiating and recapitulating important antiviral effects of IFN.

Identification of a Class of HCV Inhibitors Directed Against the Nonstructural Protein NS4B

An activity identified in hepatitis C virus NS4B paves the way for a distinct new class of antivirals. Hepatitis C is a surreptitious infection leading to inflammation of the liver, chronic liver disease, and ultimately causing cirrhosis. Nearly 150 million infected patients worldwide are in serious need of an alternative to viral suppressants in current use that have significant toxicities and are often ineffective. Cho and colleagues now take a closer look at the molecular virology of hepatitis C and identify a crucial new function for a largely uncharacterized protein, NS4B. They found that a particular region of this protein, which is critically involved in forming small vesicle aggregates that form the hypothesized platform to facilitate viral genome replication, can be manipulated to serve as a readout for high-throughput screens aimed at uncovering small-molecule pharmacological inhibitors of hepatitis C genome replication. Screening through a milieu of possibilities, they demonstrate the utility of two such compounds and tease apart the mechanism and biochemical activities by which these small inhibitors disrupt NS4B function and ultimately viral replication. Whether these new compounds can be used as monotherapies or in amalgamation with current strategies awaits further testing. New classes of drugs are needed to combat hepatitis C virus (HCV), an important worldwide cause of liver disease. We describe an activity of a key domain, an amphipathic helix we termed 4BAH2, within a specific HCV nonstructural protein, NS4B. In addition to its proposed role in viral replication, we validate 4BAH2 as essential for HCV genome replication and identify first-generation small-molecule inhibitors of 4BAH2 that specifically prevent HCV replication within cells. Mechanistic studies reveal that the inhibitors target 4BAH2 function by preventing either 4BAH2 oligomerization or 4BAH2 membrane association. 4BAH2 inhibitors represent an additional class of compounds with potential to effectively treat HCV.

Potential for Hepatitis C Virus Resistance to Nitazoxanide or Tizoxanide

ABSTRACT Nitazoxanide and its primary metabolite, tizoxanide, inhibit hepatitis C virus (HCV) replication in HCV replicon systems. To study the potential for resistance, we subjected Huh7 cells harboring HCV replicons to serial passage in 250 μM G418 and increasing concentrations of nitazoxanide or tizoxanide. Passage of the replicon-containing cell lines in either compound resulted in increases in the 50% effective concentrations (EC50s) (7- to 13-fold), EC90s (14- to 36-fold), and 50% cytotoxic concentrations (2- to 4-fold) of both compounds. Serial passage in either compound did not alter the susceptibility of HCV replicons to ribavirin or 2′-C-methylcytidine. Interestingly, serial passage in nitazoxanide or tizoxanide resulted in increased sensitivity to alpha interferon 2b: EC50s and EC90s were reduced three- and eightfold, respectively. Replicons isolated from these cell lines had no greater ability to confer tizoxanide resistance, or increased susceptibility to alpha interferon, than replicons isolated from the parental cell line that had not previously been exposed to nitazoxanide or tizoxanide. These findings are indicative of a cell-mediated activity differing from that of other anti-HCV drugs but complementary with interferon and are consistent with the enhanced response rates observed clinically when nitazoxanide is combined with pegylated interferon therapy. Finally, unlike data for other compounds in advanced clinical development for HCV, these data are consistent with resistance in HCV replicon-containing cell lines conferred by changes in the host and not by mutations in the virus.

Isolation and Transcriptional Profiling of Purified Hepatic Cells Derived from Human Embryonic Stem Cells

The differentiation of human embryonic stem cells (hESCs) into functional hepatocytes provides a powerful in vitro model system for studying the molecular mechanisms governing liver development. Furthermore, a well‐characterized renewable supply of hepatocytes differentiated from hESCs could be used for in vitro assays of drug metabolism and toxicology, screening of potential antiviral agents, and cell‐based therapies to treat liver disease. In this study, we describe a protocol for the differentiation of hESCs toward hepatic cells with complex cellular morphologies. Putative hepatic cells were identified and isolated using a lentiviral vector, containing the α‐fetoprotein promoter driving enhanced green fluorescent protein expression (AFP:eGFP). Whole‐genome transcriptional profiling was performed on triplicate samples of AFP:eGFP+ and AFP:eGFP− cell populations using the recently released Affymetrix Exon Array ST 1.0 (Santa Clara, CA, http://www.affymetrix.com). Statistical analysis of the transcriptional profiles demonstrated that the AFP:eGFP+ population is highly enriched for genes characteristic of hepatic cells. These data provide a unique insight into the complex process of hepatocyte differentiation, point to signaling pathways that may be manipulated to more efficiently direct the differentiation of hESCs toward mature hepatocytes, and identify molecular markers that may be used for further dissection of hepatic cell differentiation from hESCs.