PubMed Health⌕ Search

Biomedical subjects

Darius Moradpour

Publications and source records attributed to Darius Moradpour.

At least 19 recordsLinked to original sources
PubMed2006

Conserved determinants for membrane association of nonstructural protein 5A from hepatitis C virus and related viruses.

Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.

Amino Acid Sequence↗
PubMed2006

euHCVdb: the European hepatitis C virus database.

The hepatitis C virus (HCV) genome shows remarkable sequence variability, leading to the classification of at least six major genotypes, numerous subtypes and a myriad of quasispecies within a given host. A database allowing researchers to investigate the genetic and structural variability of all available HCV sequences is an essential tool for studies on the molecular virology and pathogenesis of hepatitis C as well as drug design and vaccine development. We describe here the European Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr), a collection of computer-annotated sequences based on reference genomes. The annotations include genome mapping of sequences, use of recommended nomenclature, subtyping as well as three-dimensional (3D) molecular models of proteins. A WWW interface has been developed to facilitate database searches and the export of data for sequence and structure analyses. As part of an international collaborative effort with the US and Japanese databases, the European HCV Database (euHCVdb) is mainly dedicated to HCV protein sequences, 3D structures and functional analyses.

Databases, Protein↗
PubMed2006

Molecular virology of hepatitis C virus (HCV): 2006 update.

Fascinating progress in the understanding of the molecular biology of hepatitis C virus (HCV) was achieved recently. The replicon system revolutionized the investigation of HCV RNA replication and facilitated drug discovery. Novel systems for functional analyses of the HCV glycoproteins allowed the validation of HCV receptor candidates and the investigation of cell entry mechanisms. Most recently, recombinant infectious HCV could be produced in cell culture, rendering all steps of the viral life cycle, including entry and release of viral particles, amenable to systematic analysis. In this review, we summarize recent advances and discuss future research directions.

Journal Article↗
PubMed2006

NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a monotopic membrane protein anchored to the membrane by an N-terminal in-plane amphipathic alpha-helix. This membrane anchor is essential for the assembly of a functional viral replication complex. Although amino acid sequences differ considerably, putative membrane anchors with amphipathic features were predicted in NS5A from related Flaviviridae family members, in particular bovine viral diarrhea virus (BVDV), the prototype representative of the genus Pestivirus. We report here the NMR structure of the membrane anchor 1-28 of NS5A from BVDV in the presence of different membrane mimetic media. This anchor includes a long amphipathic alpha-helix of 21 residues interacting in-plane with the membrane interface and including a putative flexible region. Molecular dynamic simulation at a water-dodecane interface used to mimic the surface separating a lipid bilayer and an aqueous medium demonstrated the stability of the helix orientation and the location at the hydrophobic-hydrophilic interface. The flexible region of the helix appears to be required to allow the most favorable interaction of hydrophobic and hydrophilic side chain residues with their respective environment at the membrane interface. Despite the lack of amino acid sequence similarity, this amphipathic helix shares common structural features with that of the HCV counterpart, including a stable, hydrophobic N-terminal segment separated from the more hydrophilic C-terminal segment by a local, flexible region. These structural conservations point toward conserved roles of the N-terminal in-plane membrane anchors of NS5A in replication complex formation of HCV, BVDV, and other related viruses.

Amino Acid Sequence↗
PubMed2005

Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.

Antiviral immunity against a pathogen is mounted upon recognition by the host of virally associated structures. One of these viral 'signatures', double-stranded (ds) RNA, is a replication product of most viruses within infected cells and is sensed by Toll-like receptor 3 (TLR3) and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I, also known as Ddx58) and Mda5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard). Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-kappaB. Here we describe Cardif, a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKalpha, IKKbeta and IKKvarepsilon kinases by means of its C-terminal region, leading to the activation of NF-kappaB and IRF3. Overexpression of Cardif results in interferon-beta and NF-kappaB promoter activation, and knockdown of Cardif by short interfering RNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from hepatitis C virus known to block interferon-beta production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes.

Adaptor Proteins, Signal Transducing↗
PubMed2005

[Hepatocellular carcinoma: a multidisciplinary approach].

The treatment of the hepatocellular carcinoma (HCC) is multidisciplinary. Hepatic transplantation offers the best chances of survival for patients with hepatic cirrhosis and HCC. However the indications for transplantation are limited. For patients that do not qualify for liver transplantation, surgical excision and percutaneous ablative treatment can also be curative. Five years survival then reaches 50%. The choice of treatment is based upon the patient's clinical state, the hepatic function and the cancer clinical stage. Follow-up is crucial as recurrences can be treated by following similar algorithms. The efficacy of oncological adjuvant and neoadjuvant treatment is not yet proved.

Algorithms↗
PubMed2005

Function follows form: the structure of the N-terminal domain of HCV NS5A.

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The nonstructural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kd) with an amphipathic -helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into 3 domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif, and a disulfide bond. We use molecular surface analysis to suggest the location of protein-, RNA-, and membrane-interaction sites.

Binding Sites↗
PubMed2005

Pathogenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide. The major aetiologies and risk factors for the development of HCC are well defined and some of the multiple steps involved in hepatocarcinogenesis have been elucidated in recent years. However, no clear picture of how and in what sequence these factors interact at the molecular level has emerged yet. Malignant transformation of hepatocytes may occur as a consequence of various aetiologies, such as chronic viral hepatitis, alcohol, and metabolic disorders, in the context of increased cellular turnover induced by chronic liver injury, regeneration and cirrhosis. Activation of cellular oncogenes, inactivation of tumour suppressor genes, genomic instability, including DNA mismatch repair defects and impaired chromosomal segregation, overexpression of growth and angiogenic factors, and telomerase activation may contribute to the development of HCC. Overall, HCCs are genetically very heterogeneous tumours. New technologies, including gene expression profiling and proteomic analyses, should allow us to further elucidate the molecular events underlying HCC development and identify novel diagnostic markers as well as therapeutic targets.

Carcinoma, Hepatocellular↗
PubMed2005

Stable human lymphoblastoid cell lines constitutively expressing hepatitis C virus proteins.

The cellular immune response plays a central role in virus clearance and pathogenesis of liver disease in hepatitis C. The study of hepatitis C virus (HCV)-specific immune responses is limited by currently available cell-culture systems. Here, the establishment and characterization of stable human HLA-A2-positive B-lymphoblastoid x T hybrid cell lines constitutively expressing either the NS3-4A complex or the entire HCV polyprotein are reported. These cell lines, termed T1/NS3-4A and T1/HCVcon, respectively, were maintained in continuous culture for more than 1 year with stable characteristics. HCV structural and non-structural proteins were processed accurately, indicating that the cellular and viral proteolytic machineries are functional in these cell lines. Viral proteins were found in the cytoplasm in dot-like structures when expressed in the context of the HCV polyprotein or in a perinuclear fringe when the NS3-4A complex was expressed alone. T1/NS3-4A and T1/HCVcon cells were lysed efficiently by HCV-specific cytotoxic T lymphocytes from patients with hepatitis C and from human HLA-A2.1 transgenic mice immunized with a liposomal HCV vaccine, indicating that viral proteins are processed endogenously and presented efficiently via the major histocompatibility complex class I pathway. In conclusion, these cell lines represent a unique tool to study the cellular immune response, as well as to evaluate novel vaccine and immunotherapeutic strategies against HCV.

Animals↗
PubMed2005

Characterization of nonstructural protein membrane anchor deletion mutants expressed in the context of the hepatitis C virus polyprotein.

Protein-protein interactions involved in formation of the membrane-associated hepatitis C virus (HCV) replication complex are poorly understood. Here, we investigated nonstructural proteins with deletions in their membrane anchor domains when expressed in the context of the entire HCV polyprotein. Interactions among cytosolic domains of HCV nonstructural proteins were found not to be sufficiently strong to rescue such mutants to the membrane. Thus, the membrane anchor domains of nonstructural proteins are essential for incorporation of these proteins into the HCV replication complex while interactions among the cytosolic domains appear to be relatively weak. This feature may provide the nonstructural proteins with a certain flexibility to perform their multiple functions during HCV replication.

Cell Line, Tumor↗
PubMed2005

Upregulation of protein phosphatase 2Ac by hepatitis C virus modulates NS3 helicase activity through inhibition of protein arginine methyltransferase 1.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. HCV has a positive-strand RNA genome of about 9.4 kb in size, which serves as a template for replication and for translation of a polyprotein of about 3,000 amino acids. The polyprotein is cleaved co- and posttranslationally by cellular and viral proteases into at least 10 different mature proteins. One of these proteins, nonstructural protein 3 (NS3), has serine protease and NTPase/RNA helicase activity. Arginine 467 in the helicase domain of NS3 (arginine 1493 in the polyprotein) can be methylated by protein arginine methyltransferase 1 (PRMT1). Here we report that the methylation of NS3 inhibits the enzymatic activity of the helicase. Furthermore, we found that PRMT1 activity itself is regulated by protein phosphatase 2A (PP2A). PP2A inhibits PRMT1 enzymatic activity and therefore increases the helicase activity of NS3. This is important, because we found an increased expression of PP2A in cell lines with inducible HCV protein expression, in transgenic mice expressing HCV proteins in hepatocytes, and in liver biopsy samples from patients with chronic hepatitis C. Interestingly, up-regulation of PP2A not only modulates the enzymatic activity of an important viral protein, NS3 helicase, but also interferes with the cellular defense against viruses by inhibiting interferon-induced signaling through signal transducer and activator of transcription 1 (STAT1). We conclude that up-regulation of PP2A might be crucial for the efficient replication of HCV and propose PP2A as a potential target for anti-HCV treatment strategies.

Cell Line↗
PubMed2004

A liposomal peptide vaccine inducing CD8+ T cells in HLA-A2.1 transgenic mice, which recognise human cells encoding hepatitis C virus (HCV) proteins.

Virus specific T cell responses play an important role in resolving acute hepatitis C virus (HCV) infections. Using the HLA-A2.1 transgenic mouse model we investigated the potential of a liposomal peptide vaccine to prime a CD8(+) T cell response against 10 different HCV epitopes, relevant for human applications. We were able to demonstrate the induction of strong cytotoxic T cell responses and high numbers of IFN-gamma-secreting cells, which persisted at high levels for at least 3 months. Co-integrating CpG oligonucleotides into liposomes further increased the number of IFN-gamma-secreting cells by 2-10-fold for most epitopes tested. The frequency of specific cells was further analysed with chimeric A2 tetramers bearing the NS31073-1081 epitope and was estimated at 2-23% of the CD8(+) T cell population. Importantly, mouse effector cells, specific for this epitope, were also capable of lysing a human target cell line expressing HCV proteins. This finding and the specific protection observed in challenge experiments with recombinant vaccinia virus expressing HCV sequences emphasise the biological relevance of the vaccine-induced immune response. In conclusion, such liposome formulations represent a safe and promising strategy to stimulate the CD8(+) T cell against HCV.

Animals↗
PubMed2004

Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane anchor domain of NS5A as determined by NMR spectroscopy. An alpha-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic alpha-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.

Amino Acid Sequence↗
PubMed2004

Structural biology of hepatitis C virus.

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.

Hepacivirus↗
PubMed2004

A primer on the molecular virology of hepatitis C.

Exciting advances have recently been made in the understanding of the molecular virology of hepatitis C. Powerful model systems have been developed that allow to systematically dissect important steps of the hepatitis C virus (HCV) life cycle. These include new systems for functional analyses of the HCV glycoproteins, providing insights into possible HCV receptors and cell entry mechanisms, and the replicon system, which has revolutionized investigation of HCV RNA replication and has facilitated drug discovery efforts. The largest gaps remain in the understanding of the virion structure and the processes that lead to the assembly, packaging and release of virions. However, given the pace of current HCV research, progress in these directions may be expected in the near future. Here, we provide a primer on the molecular virology of hepatitis C, with particular reference to novel antiviral targets and therapeutic strategies.

Animals↗