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Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

International audience; Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a RNA-binding phosphoprotein composed of a N-terminal membrane anchor (AH), a structured domain 1 (D1), and two intrinsically disordered domains (D2 and D3). The knowledge of the functional architecture of this multifunctional protein remains limited. We report here that NS5A-D1D2D3 produced in a wheat germ cell-free system is obtained under a highly phosphorylated state. Its NMR analysis revealed that these phosphorylations do not change the disordered nature of D2 and D3 domains but increase the number of conformers due to partial phosphorylations. By combining NMR and small angle X-ray scattering, we performed a comparative structural characterization of unphosphorylated recombinant D2 domains of JFH1 (genotype 2a) and the Con1 (genotype 1b) strains produced in Escherichia coli. These analyses highlighted a higher intrinsic folding of the latter, revealing the variability of intrinsic conformations in HCV genotypes. We also investigated the effect of D2 mutations conferring resistance of HCV replication to cyclophilin A (CypA) inhibitors on the structure of the recombinant D2 Con1 mutants and their binding to CypA. Although resistance mutations D320E and R318W could induce some local and/or global folding perturbation, which could thus affect the kinetics of conformer interconversions, they do not significantly affect the kinetics of CypA/D2 interaction measured by surface plasmon resonance (SPR). The combination of all our data led us to build a model of the overall structure of NS5A, which provides a useful template for further investigations of the structural and functional features of this enigmatic protein.

The viroporin p7 of the hepatitis C virus forms multimeric channels eligible for ion transport across the endoplasmic reticulum membrane. Currently the subject of many studies and discussion, the molecular assembly of the ion channel and the structural characteristics of the p7 monomer are not yet fully understood. Structural investigation of p7 has been carried out only in detergent environments, making the interpretation of the experimental results somewhat questionable. Here, we analyze by means of molecular dynamics simulations the structure of the p7 monomer as a function of its sequence, initial conformation and environment. We investigate the conductance properties of three models of a hexameric p7 ion channel by examining ion translocation in a pure lipid bilayer. It is noteworthy that although none of the models reflects the experimentally observed trend to conduct preferentially cations, we were able to identify the position and orientation of titratable acidic or basic residues playing a crucial role in ion selectivity and in the overall conductance of the channel. In addition, too compact a packing of the monomers leads to channel collapse rather than formation of a reasonable pore, amenable to ion translocation. The present findings are envisioned to help assess the physiological relevance of p7 ion channel models consisting of multimeric structures obtained in non-native environments.

Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle., Author summary Despite remarkable progress in the development of therapeutic options, more than 70 million individuals are chronically infected by hepatitis C virus (HCV) worldwide and major challenges in basic and translational research remain. Phylogenetically-related HCV homologues have recently been identified in the wild in several mammalian species, whose host restriction and potential for zoonosis remain largely unknown. We comparatively characterized the functions and properties of nonstructural proteins 2 (NS2) from several animal hepaciviruses and HCV. We demonstrated that NS2 from animal hepaciviruses, like HCV NS2, are cysteine proteases, which function as dimers with two composite active sites to ensure a key proteolytic event of the single viral polyprotein at the NS2/NS3 junction. In addition to the activation of HCV NS2 protease by NS3 N-terminal domain, our data revealed a novel NS3-independent substrate specificity and efficient intrinsic proteolytic activity of NS2. The conservation of its properties and peculiar mode of action among distantly related hepaciviruses supports an important regulatory role for NS2 protein in the life cycle of these viruses. It also strengthens the value of animal, notably rodent hepaciviruses for the development of surrogate, immunocompetent models of HCV infection to address HCV-associated pathogenesis and vaccine strategies.

In hepatitis C virus (HCV)-infected cells, the envelope glycoproteins E1 and E2 assemble as a heterodimer. To investigate potential changes in the oligomerization of virion-associated envelope proteins, we performed SDS-PAGE under reducing conditions but without thermal denaturation. This revealed the presence of SDS-resistant trimers of E1 in the context of cell-cultured HCV (HCVcc) as well as in the context of HCV pseudoparticles (HCVpp). The formation of E1 trimers was found to depend on the coexpression of E2. To further understand the origin of E1 trimer formation, we coexpressed in bacteria the transmembrane (TM) domains of E1 (TME1) and E2 (TME2) fused to reporter proteins and analyzed the fusion proteins by SDS-PAGE and Western blotting. As expected for strongly interacting TM domains, TME1–TME2 heterodimers resistant to SDS were observed. These analyses also revealed homodimers and homotrimers of TME1, indicating that such complexes are stable species. The N-terminal segment of TME1 exhibits a highly conserved GxxxG sequence, a motif that is well documented to be involved in intramembrane protein-protein interactions. Single or double mutations of the glycine residues (Gly354 and Gly358) in this motif markedly decreased or abrogated the formation of TME1 homotrimers in bacteria, as well as homotrimers of E1 in both HCVpp and HCVcc systems. A concomitant loss of infectivity was observed, indicating that the trimeric form of E1 is essential for virus infectivity. Taken together, these results indicate that E1E2 heterodimers form trimers on HCV particles, and they support the hypothesis that E1 could be a fusion protein. IMPORTANCE HCV glycoproteins E1 and E2 play an essential role in virus entry into liver cells as well as in virion morphogenesis. In infected cells, these two proteins form a complex in which E2 interacts with cellular receptors, whereas the function of E1 remains poorly understood. However, recent structural data suggest that E1 could be the protein responsible for the process of fusion between viral and cellular membranes. Here we investigated the oligomeric state of HCV envelope glycoproteins. We demonstrate that E1 forms functional trimers after virion assembly and that in addition to the requirement for E2, a determinant for this oligomerization is present in a conserved GxxxG motif located within the E1 transmembrane domain. Taken together, these results indicate that a rearrangement of E1E2 heterodimer complexes likely occurs during the assembly of HCV particles to yield a trimeric form of the E1E2 heterodimer. Gaining structural information on this trimer will be helpful for the design of an anti-HCV vaccine.

ABSTRACT HCV encodes two envelope glycoproteins, E1 and E2, which assemble as a non-covalent heterodimer in infected cells. During HCV morphogenesis, these proteins are incorporated into viral particles and they are the major viral determinants of HCV entry. Functional studies have revealed unique features in these viral envelope glycoproteins. Indeed, E1–E2 interaction, mediated by their transmembrane domain, is essential for HCV assembly and entry. Furthermore, recent data also show that these glycoproteins interact with apolipoproteins. Recent crystallography data provide some structural support to better understand how these proteins interact with the host. In this review, we summarize the biogenesis of HCV envelope glycoproteins and their role in HCV morphogenesis in the context of the hijacking of the very low-density lipoprotein assembly pathway by this virus. We also describe the functions of HCV glycoproteins during virus entry with a special focus on the unexpected structural features of E2 glycoprotein. Finally, we discuss the major neutralizing epitopes in the light of E2 structure.

Affiliations ECOFECT; International audience; Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural proteins 2, 4B and 5A membrane proteins of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

Due to their hydrophobic nature, membrane proteins are notoriously difficult to express in classical cell-based protein expression systems. Often toxic, they also undergo degradation in cells or aggregate in inclusion bodies, making delicate issues further solubilization and renaturation. These are major bottlenecks in their structural and functional analysis. The wheat germ cell-free (WGE-CF) system offers an effective alternative not only to classical cell-based protein expression systems but also to other cell-free systems for the expression of membrane proteins. The WGE-CF indeed allows the production of milligram amounts of membrane proteins in a detergent-solubilized, homogenous, and active form. Here, we describe the method to produce a viral integral membrane protein, which is the non-structural protein 2 (NS2) of hepatitis C virus, in view of structural studies by solid-state NMR in a native-like lipid environment.

Due to their hydrophobic nature, membrane proteins are notoriously difficult to express in classical cell-based protein expression systems. Often toxic, they also undergo degradation in cells or aggregate in inclusion bodies, making delicate issues further solubilization and renaturation. These are major bottlenecks in their structural and functional analysis. The wheat germ cell-free (WGE-CF) system offers an effective alternative not only to classical cell-based protein expression systems but also to other cell-free systems for the expression of membrane proteins. The WGE-CF indeed allows the production of milligram amounts of membrane proteins in a detergent-solubilized, homogenous, and active form. Here, we describe the method to produce a viral integral membrane protein, which is the non-structural protein 2 (NS2) of hepatitis C virus, in view of structural studies by solid-state NMR in a native-like lipid environment.

The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.

We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [(2)H,(13)C,(15)N]-labeled protein are shown to yield narrow (13)C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

International audience; Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.

International audience; Insertions or deletions (indels) of amino acids residues have been recognized as an important source of genetic and structural divergence between paralogous Bcl-2 family members. However, these signature sequences have not so far been extensively investigated amongst orthologous Bcl-2 family proteins. Bcl2l10 is an antiapoptotic member of the Bcl-2 family that has evolved rapidly throughout the vertebrate lineage and which shows conserved abundant expression in eggs and oocytes. In this paper, we have unraveled two major sites of divergence between human Bcl2l10 and its vertebrate homologs. The first one provides length variation at the N-terminus (before the BH4 domain) and the second one is located between the predicted α5-α6 pore-forming helices, providing an unprecedented case in the superfamily of helix-bundled pore-forming proteins. These two particular indels were studied phylogenetically and through biochemical and cell biological techniques, including truncation and site-directed mutagenesis. While deletion of the N-terminal extension had no significant functional impact in HeLa cells, our results suggest that the human Bcl2l10 protein evolved a calcium-binding motif in its α5-α6 interhelical region by acquiring critical negatively charged residues. Considering the reliance of female eggs on calcium-dependent proteins and calcium-regulated processes and the exceptional longevity of oocytes in the primate lineage, we propose that this microstructural variation may be an adaptive feature associated with high maternal expression of this Bcl-2 family member.

International audience; The expression of soluble, functional protein on a preparative scale poses a central challenge for structural studies. Cell-free protein expression has become a valuable alternative to cell-based methods, and allows today the expression of milligram quantities of protein. Its use is particularly attractive for NMR studies as it allows a multitude of isotopic labeling schemes. We have implemented and further developed protocols to prepare cell-free extracts from wheat germ to produce recombinant protein for solid-state NMR studies. Furthermore, we established the Renilla luciferase model to optimise and evaluate extract quality, and report first productions of the prions Ure2p and HET-s devoted to structural studies currently ongoing in our laboratories.

Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic α-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored protein with a highly conserved C-terminal transmembrane domain (TMD) that is required for the assembly of a functional replication complex. Here, we report that the TMD of the HCV RdRp can be functionally replaced by a newly identified analogous membrane anchor of the GB virus B (GBV-B) NS5B RdRp. Replicons with a chimeric RdRp consisting of the HCV catalytic domain and the GBV-B membrane anchor replicated with reduced efficiency. Compensatory amino acid changes at defined positions within the TMD improved the replication efficiency of these chimeras. These observations highlight a conserved structural motif within the TMD of the HCV NS5B RdRp that is required for RNA replication.

Hepatitis C virus (HCV) is a positive-strand RNA virus that replicates its genome in a membrane-associated replication complex. Nonstructural protein 4B (NS4B) induces the specific membrane alteration, designated as membranous web (MW), that harbours this complex. HCV NS4B is an integral membrane protein predicted to comprise four transmembrane segments in its central part. The N-terminal part comprises two amphipathic alpha-helices of which the second has the potential to traverse the membrane bilayer, likely upon oligomerisation. The C-terminal part comprises a predicted highly conserved alpha-helix, a membrane-associated amphipathic alpha-helix and two reported palmitoylation sites. NS4B interacts with other viral nonstructural proteins and has been reported to bind viral RNA. In addition, it was found to harbour an NTPase activity. Finally, NS4B has recently been found to have a role in viral assembly. Much work needs to be done with respect to further dissecting these multiple functions as well as providing a refined membrane topology and complete structure of NS4B. Progress in this direction should yield important insights into the functional architecture of the HCV replication complex and may reveal new opportunities for antiviral intervention against a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.

Hepatitis C virus (HCV) p7 is an integral membrane protein that forms ion channels in vitro and that is crucial for the efficient assembly and release of infectious virions. Due to these properties, p7 was included in the family of viroporins that comprises proteins like influenza A virus M2 and human immunodeficiency virus type 1 (HIV-1) vpu, which alter membrane permeability and facilitate the release of infectious viruses. p7 from different HCV isolates sustains virus production with variable efficiency. Moreover, p7 determinants modulate processing at the E2/p7 and the p7/NS2 signal peptidase cleavage sites, and E2/p7 cleavage is incomplete. Consequently, it was unclear if a differential ability to sustain virus production was due to variable ion channel activity or due to alternate processing at these sites. Therefore, we developed a trans -complementation assay permitting the analysis of p7 outside of the HCV polyprotein and thus independently of processing. The rescue of p7-defective HCV genomes was accomplished by providing E2, p7, and NS2, or, in some cases, by p7 alone both in a transient complementation assay as well as in stable cell lines. In contrast, neither influenza A virus M2 nor HIV-1 vpu compensated for defective p7 in HCV morphogenesis. Thus, p7 is absolutely essential for the production of infectious HCV particles. Moreover, our data indicate that p7 can operate independently of an upstream signal sequence, and that a tyrosine residue close to the conserved dibasic motif of p7 is important for optimal virus production in the context of genotype 2a viruses. The experimental system described here should be helpful to investigate further key determinants of p7 that are essential for its structure and function in the absence of secondary effects caused by altered polyprotein processing.

International audience; We report a biochemical and structural characterization of domain 2 of the non-structural 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 (CsA) 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 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.

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-κB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-κB in antiviral innate immunity.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic α-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic α-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

The hepatitis C virus (HCV) is a flavivirus replicating in the cytoplasm of infected cells. The HCV genome is a single-stranded RNA encoding a polyprotein that is cleaved by cellular and viral proteases into 10 different products. While the structural proteins core protein, envelope protein 1 (E1) and E2 build up the virus particle, most nonstructural (NS) proteins are required for RNA replication. One of the least studied proteins is NS2, which is composed of a C-terminal cytosolic protease domain and a highly hydrophobic N-terminal domain. It is assumed that the latter is composed of three trans-membrane segments (TMS) that tightly attach NS2 to intracellular membranes. Taking advantage of a system to study HCV assembly in a hepatoma cell line, in this study we performed a detailed characterization of NS2 with respect to its role for virus particle assembly. In agreement with an earlier report (Jones, C. T., Murray, C. L., Eastman, D. K., Tassello, J., and Rice, C. M. (2007) J. Virol.81 ,8374 -838317537845), we demonstrate that the protease domain, but not its enzymatic activity, is required for infectious virus production. We also show that serine residue 168 in NS2, implicated in the phosphorylation and stability of this protein, is dispensable for virion formation. In addition, we determined the NMR structure of the first TMS of NS2 and show that the N-terminal segment (amino acids 3-11) forms a putative flexible helical element connected to a stable α-helix (amino acids 12-21) that includes an absolutely conserved helix side in genotype 1b. By using this structure as well as the amino acid conservation as a guide for a functional study, we determined the contribution of individual amino acid residues in TMS1 for HCV assembly. We identified several residues that are critical for virion formation, most notably a central glycine residue at position 10 of TMS1. Finally, we demonstrate that mutations in NS2 blocking HCV assembly can be rescued by trans-complementation.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane α-helix that may be involved in intramembrane protein–protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix α 0 , formed by NS3 residues 12–23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.

Hepatitis C virus (HCV) afflicts more than 170 million people worldwide causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The recent development of complete cell-culture systems for HCV has accelerated the pace of hepatitis research. Specifically, these techniques have provided new insights into the virus lifecycle that are reviewed here. This should pave the way for developing bespoke and effective antiviral therapies and vaccines. Exciting progress has recently been made in understanding the replication of hepatitis C virus, a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. The development of complete cell-culture systems should now enable the systematic dissection of the entire viral lifecycle, providing insights into the hitherto difficult-to-study early and late steps. These efforts have already translated into the identification of novel antiviral targets and the development of new therapeutic strategies, some of which are currently undergoing clinical evaluation.

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.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro314–Trp316 turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro314 of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. 290;31

Non-structural protein 2 (NS2) of the hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease and that plays a central organizing role in assembly of infectious progeny virions. While the crystal structure of the protease domain has been solved, the NS2 full-length form remains biochemically and structurally uncharacterized because recombinant NS2 could not be prepared in sufficient quantities from cell-based systems. We show here that functional NS2 in the context of the NS2-NS3pro precursor protein, ensuring NS2-NS3 cleavage, can be efficiently expressed by using a wheat germ cell-free expression system. In this same system, we subsequently successfully produce and purify milligram amounts of a detergent-solubilized form of full-length NS2 exhibiting the expected secondary structure content. Furthermore, immuno-electron microscopy analyses of reconstituted proteoliposomes demonstrate NS2 association with model membranes.

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

International audience; It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.It has been suggested that the hepatitis C virus (HCV) infects host cells through a pH-dependent internalization mechanism, but the steps leading from virus attachment to the fusion of viral and cellular membranes remain uncharacterized. Here we studied the mechanism underlying the HCV fusion process in vitro using liposomes and our recently described HCV pseudoparticles (pp) bearing functional E1E2 envelope glycoproteins. The fusion of HCVpp with liposomes was monitored with fluorescent probes incorporated into either the HCVpp or the liposomes. To validate these assays, pseudoparticles bearing either the hemagglutinin of the influenza virus or the amphotropic glycoprotein of murine leukemia virus were used as models for pH-dependent and pH-independent entry, respectively. The use of assays based either on fusion-induced dequenching of fluorescent probes or on reporter systems, which produce fluorescence when the virus and liposome contents are mixed, allowed us to demonstrate that HCVpp mediated a complete fusion process, leading to the merging of both membrane leaflets and to the mixing of the internal contents of pseudoparticle and liposome. This HCVpp-mediated fusion was dependent on low pH, with a threshold of 6.3 and an optimum at about 5.5. Fusion was temperature-dependent and did not require any protein or receptor at the surface of the target liposomes. Most interestingly, fusion was facilitated by the presence of cholesterol in the target membrane. These findings clearly indicate that HCV infection is mediated by a pH-dependent membrane fusion process. This paves the way for future studies of the mechanisms underlying HCV membrane fusion.

Hepatitis C virus (HCV) E1 and E2 envelope glycoproteins (GPs) displayed on retroviral cores (HCVpp) are a powerful and highly versatile model system to investigate wild-type HCV entry. To further characterize this model system, the cellular site of HCVpp assembly and the respective roles of the HCV GPs in this process were investigated. By using a combination of biochemical methods with confocal and electron microscopic techniques, it was shown that, in cells producing HCVpp, both E1 and E2 colocalized with retroviral core proteins intracellularly, presumably in multivesicular bodies, but not at the cell surface. When E1 and E2 were expressed individually with retroviral core proteins, only E2 colocalized with and was incorporated on retroviral cores. Conversely, the colocalization of E1 with retroviral core proteins and its efficient incorporation occurred only upon co-expression of E2. Moreover, HCVpp infectivity correlated strictly with the presence of both E1 and E2 on retroviral cores. Altogether, these results confirm that the E1E2 heterodimer constitutes the prebudding form of functional HCV GPs and, more specifically, show that dimerization with E2 is a prerequisite for efficient E1 incorporation onto particles.

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.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.

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.

The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because approximately 10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVDeltaEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle.The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because approximately 10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVDeltaEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle.

International audience; The crystal structure of the regulatory protein Crh from Bacillus subtilis was solved at 1.8A resolution and showed an intertwined dimer formed by N-terminal beta1-strand swapping of two monomers. Comparison with the monomeric NMR structure of Crh revealed a domain swap induced conformational rearrangement of the putative interaction site with the repressor CcpA. The resulting conformation closely resembles that observed for the monomeric Crh homologue HPr, indicating that the Crh dimer is the active form binding to CcpA. An analogous dimer of HPr can be constructed without domain swapping, suggesting that HPr may dimerize upon binding to CcpA. Our data suggest that reversible 3D domain swapping of Crh might be an efficient regulatory mechanism to modulate its activity.The crystal structure of the regulatory protein Crh from Bacillus subtilis was solved at 1.8A resolution and showed an intertwined dimer formed by N-terminal beta1-strand swapping of two monomers. Comparison with the monomeric NMR structure of Crh revealed a domain swap induced conformational rearrangement of the putative interaction site with the repressor CcpA. The resulting conformation closely resembles that observed for the monomeric Crh homologue HPr, indicating that the Crh dimer is the active form binding to CcpA. An analogous dimer of HPr can be constructed without domain swapping, suggesting that HPr may dimerize upon binding to CcpA. Our data suggest that reversible 3D domain swapping of Crh might be an efficient regulatory mechanism to modulate its activity.