euHCVdb: the European hepatitis C virus database

Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can be triggered in a receptor-independent but pH-dependent manner and that fusion of the HCV particles with liposomes is dependent on the viral dose and on the lipid composition of the target membranes. In addition CBH-5, an HCV E2-specific antibody, inhibited fusion in a dose-dependent manner. Interestingly, point mutations in E2, known to abrogate HCV glycoprotein-mediated fusion in a cell-based assay, altered or even abolished fusion in the liposome-based assay. When assaying the fusion properties of HCV particles with different buoyant density, we noted higher fusogenicity of particles with lower density. This could be attributable to inherently different properties of low density particles, to association of these particles with factors stimulating fusion, or to co-floatation of factors enhancing fusion activity in trans. Taken together, these data show the important role of lipids of both the viral and target membranes in HCV-mediated fusion, point to a crucial role played by the E2 glycoprotein in the process of HCV fusion, and reveal an important behavior of HCV of different densities with regard to fusion. Hepatitis C virus (HCV)4 is an important public health concern worldwide as it is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped virus that belongs to the Hepacivirus genus of the Flaviviridae family (1). Based on sequence comparison, patient isolates are classified into seven genotypes, differing in their nucleotide sequence by 30 -35% (2-5). The two viral surface proteins, E1 (residues 192-383) and E2 (residues 384 -746), are processed by signal peptidases of the endoplasmic reticulum from a 3,000-amino acid-long polyprotein encoded by the HCV genome (reviewed in Ref.2). The E1 (ϳ31 kDa) and E2 (ϳ70 kDa) proteins are glycosylated in their large amino-terminal ectodomains (6) and are anchored in the viral membrane by their carboxyl-terminal transmembrane domains. E1 and E2 form a heterodimer stabilized by noncovalent interactions. This oligomer is thought to be present at the surface of HCV particles (7) and to be involved in viral entry. Carboxyl-terminally truncated soluble E2 protein is known to specifically bind to crucial HCV entry factors like glycosaminoglycans, the tetraspanin CD81, and the scavenger receptor BI (8 -12). Thus, virus-associated E2 is likely directly involved in interactions important for virus attachment and productive infection (reviewed in Refs. 13,14).Both HCV envelope glycoproteins are the targets for virusneutralizing antibodies (7,(15)...

Section: Discussioncontrasting

confidence: 55%

Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles

Haid

Pietschmann

Pécheur

2009

“…HCV NS5A sequences were retrieved from the European HCV Database (39). Multiple sequence alignments were performed with ClustalW (40) using default parameters.…”

Section: Methodsmentioning

confidence: 99%

“…15 N-and 15 N, 13 C-Labeled NS5A-D2 (JFH1)-The synthetic sequence coding for domain 2 of the HCV NS5A protein from JFH1 strain (euHCVdb (39); GenBank TM accession number AB047639, genotype 2a) was introduced in the bacterial expression vector pT7.7 with a His 6 tag (41). The resulting recombinant domain 2 of HCV NS5A (NS5A-D2; residues 248 -341) has extra M-and -LQHHHHHH extensions at the N and C termini, respectively.…”

Section: Expression and Purification Of Nonlabeled Andmentioning

confidence: 99%

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

Hanoulle

Badillo

Wieruszeski

et al. 2009

153

196

We report here a biochemical and structural characterization of domain 2 of the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and its interactions with cyclophilins A and B (CypA and CypB). Gel filtration chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy all indicate the natively unfolded nature of this NS5A-D2 domain. Because mutations in this domain have been linked to cyclosporin A resistance, we used NMR spectroscopy to investigate potential interactions between NS5A-D2 and cellular CypA and CypB. We observed a direct molecular interaction between NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins corresponds to their active site, whereas on NS5A-D2, it proved to be distributed over the many proline residues of the domain. NMR heteronuclear exchange spectroscopy yielded direct evidence that many proline residues in NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl cis/trans isomerase (PPIase) activity of CypA and CypB.

“…Sequence Analyses-Sequence analyses were performed using the European HCV data base (euHCVdb) website facilities at the Institute de Biologie et Chimie des Protéines (18). Multiple sequence alignments and amino acid conservation were carried out with the Clustal W program using default parameters (19).…”

Section: Methodsmentioning

confidence: 99%

The Lipid Droplet Binding Domain of Hepatitis C Virus Core Protein Is a Major Determinant for Efficient Virus Assembly

Shavinskaya

Boulant

Pénin

et al. 2007

227

231

Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6-and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly. HCV3 infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. About 170 million individuals worldwide are currently infected with HCV (1). A protective vaccine does not exist to date, and the therapeutic options are still limited (2). HCV has been classified in the Hepacivirinae genus within the family Flaviviridae (3). The viral genome is a 9.6-kb plus-strand RNA, which encodes a polyprotein of about 3,000 amino acids in a single open reading frame flanked at the 5Ј and 3Ј ends by nontranslated regions (NTRs). The NTRs are required for RNA translation and replication (reviewed in Refs. 4 and 5). An internal ribosome entry site resides in the 5Ј NTR, which allows expression of the polyprotein. It is co-and post-translationally processed by cellular and viral proteases to yield the mature structural and nonstructural (NS) proteins. The structural proteins include the core protein, which forms the viral capsid, and the envelope glycoproteins E1 and E2. They are separated from the NS proteins by p7, which is assumed to be a viroporin. The NS proteins include the NS2-3 autoprotease, the NS3 serine protease, a nucleotide triphosphatase/RNA helicase located in the C-terminal two-thirds of NS3, the NS4A serine-protease cofactor, NS4B, which induces membrane alterations, the RNA binding protein NS5A, and the NS5B RNA-dependent RNA polymerase (reviewed in Refs. 4 -6).The HCV core protein is a highly basic, RNA-binding protein. It is 191 aa in length and consists of three distinct domains: an N-terminal hydrophilic domain 1 (D1) formed by the first 117 aa; a hydrophobic D2 directly C-terminal of D1 and reaching to about aa 169; and a highly hydrophobic domain 3 (D3) spanning the C-terminal 20 aa, which serves as the signal peptide of the C-terminal E1 protein (7,8). This immature form of core protein...