NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains.

Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell.

Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture.

With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIα (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs).

IL-10 promotes homeostatic proliferation of human CD8(+) memory T cells and, when produced by CD1c(+) DCs, shapes naive CD8(+) T-cell priming.

IL-10 is an anti-inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8(+) CTL, IL-10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL-10 produced by human APC subsets in T-cell responses.

Hepatitis C Virus Deletion Mutants Are Found in Individuals Chronically Infected with Genotype 1 Hepatitis C Virus in Association with Age, High Viral Load and Liver Inflammatory Activity.

Hepatitis C virus (HCV) variants characterized by genomic deletions in the structural protein region have been sporadically detected in liver and serum of hepatitis C patients. These defective genomes are capable of autonomous RNA replication and are packaged into infectious viral particles in cells co-infected with the wild-type virus. The prevalence of such forms in the chronically HCV-infected population and the impact on the severity of liver disease or treatment outcome are currently unknown.

Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors.

Background & Aims: All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear.

Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking.

Background & Aims: Positive-sense RNA viruses remodel intracellular membranes to generate specialized membrane compartments for viral replication. Several RNA viruses, including poliovirus and hepatitis C virus (HCV), require phosphatidylinositol (PI) 4-kinases for their replication. However, it is not known how PI 4-kinases and their product, PI(4)P, facilitate host membrane reorganization and viral replication.

Human CD1c+ dendritic cells secrete high levels of IL-12 and potently prime cytotoxic T-cell responses.

Dendritic cells (DC) have the unique capacities to induce primary T-cell responses. In mice, CD8α(+)DC are specialized to cross-prime CD8(+) T cells and produce interleukin-12 (IL-12) that promotes cytotoxicity. Human BDCA-3(+)DC share several relevant characteristics with CD8α(+)DC, but the capacities of human DC subsets to induce CD8(+) T-cell responses are incompletely understood.

Hepatitis C virus-specific directly acting antiviral drugs.

The major targets for direct-acting antivirals (DAAs) are the NS3/4A protease, the NS5A protein, and the NS5B polymerase. The latter enzyme offers several target sites: the catalytic domain for nucleoside/nucleotide analogs and different allosteric sites for non-nucleoside inhibitors. Two protease inhibitors have already been approved and more than 40 new NS3/4A, NS5A, or NS5B inhibitors are in development pipeline.

Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase.

Synthesis and SAR of piperazinyl-N-phenylbenzamides as inhibitors of hepatitis C virus RNA replication in cell culture.

The RNA replication machinery of HCV is a multi-subunit membrane-associated complex. NS5A has emerged as an active component of HCV replicase, possibly involved in regulation of viral replication and resistance to the antiviral effect of interferon. We report here substituted piperazinyl-N-(aryl)benzamides as potent inhibitors of HCV replication exerted via modulation of the dimerization of NS5A.

NS5A phosphorylation and hyperphosphorylation.

NS5A phosphorylation can be studied in two ways: in living cells and in vitro. The former has several advantages: NS5A phosphorylation takes place in a cellular background and therefore might mimic more closely the real in vivo situation. Viral proteins and cellular kinases are in the correct cellular compartments, and dynamic processes like viral polyprotein processing and cellular signaling are in place.

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines.

Probing the elusive catalytic activity of vertebrate class IIa histone deacetylases.

It has been widely debated whether class IIa HDACs have catalytic deacetylase activity, and whether this plays any part in controlling gene expression. Herein, it has been demonstrated that class IIa HDACs isolated from mammalian cells are contaminated with other deacetylases, but can be prepared cleanly in Escherichia coli. These bacteria preparations have weak but measurable deacetylase activity.

Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors.

The hepatitis C virus encodes a single polyprotein that is processed by host and viral proteases to yield at least 10 mature viral proteins. The nonstructural (NS) protein 5A is a phosphoprotein, and experimental data indicate that the phosphorylation state of NS5A is important for the outcome of viral RNA replication. We were able to identify kinase inhibitors that specifically inhibit the formation of the hyperphosphorylated form of NS5A (p58) in cells.

The alpha isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation.

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence.

Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture.

Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication.

High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein.

The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro.

A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR.

Bacteria adapt their pattern of gene expression in response to a variety of external cues, including fluctuations in population density. This type of bacterial cell-to-cell communication is referred to as quorum-sensing. Quorum-sensing systems are present in many bacterial species and constitute a large collection of ligands and cognate receptors.

Determination of the stoichiometry of noncovalent complexes using reverse-phase high-performance liquid chromatography coupled with electrospray ion trap mass spectrometry.

An electrospray mass spectrometry-based methodology has been developed to have a fast and sensitive method for protein-cofactor stoichiometry determination. As model systems, we used two proteins which require the presence of cofactors for activity: TraR, a member of the LuxR family of quorum-sensing transcriptional regulators, which requires an acyl-homoserine lactone molecule called Agrobacterium autoinducer (AAI) as coinducer and the NS3 protease of hepatitis C virus which complexes with a NS4A cofactor peptide. Both TraR/AAI and NS3/NS4A are noncovalent complexes.

The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA.

The quorum sensing system allows bacteria to sense their cell density and initiate an altered pattern of gene expression after a sufficient quorum of cells has accumulated. In Agrobacterium tumefaciens, quorum sensing controls conjugal transfer of the tumour- inducing plasmid, responsible for plant crown gall disease. The core components of this system are the transcriptional regulator TraR and its inducing ligand N-(3-oxo-octanoyl)-L-homoserine lactone.

Subversion of cell signaling pathways by hepatitis C virus nonstructural 5A protein via interaction with Grb2 and P85 phosphatidylinositol 3-kinase.

Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways.

Crystallization and preliminary X-ray diffraction studies of the transcriptional regulator TraR bound to its cofactor and to a specific DNA sequence.

TraR is an Agrobacterium tumefaciens transcriptional regulator which binds the pheromone N-3-oxooctanoyl-L-homoserine lactone (AAI) in response to the bacterial population density. The TraR-AAI complex dimerizes and interacts with a specific 18-base-pair DNA sequence (TraBox), activating promoters containing this site. TraR was overexpressed and purified from Escherichia coli.