Search

Significance Viral hepatitis causes more deaths than tuberculosis and HIV-1 infection. Most cases are due to chronic infection with hepatitis B virus (HBV), which afflicts >250 million people. Current therapies are rarely curative, and new approaches are needed. Here, we report the discovery (by nuclear magnetic resonance) of a small molecule binder in the hydrophobic pocket in the HBV capsid. This structural element is, in an unknown manner, central in capsid envelopment. Binding of the pocket factor induces a distinct, stable conformation in the capsid, as expected for a signaling switch. This brings not only a new molecular view on the mechanism underlying capsid envelopment, but it also opens a rationale for its inhibition., Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid–envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the “synergistic double interaction” hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.

International audience; The Hepatitis C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A selfinteraction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AHlinker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.

Proton‐detected 100 kHz magic‐angle‐spinning (MAS) solid‐state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell‐free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid‐environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D 1H‐detected 1H,15N and 3D 1H,13C,15N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino‐acid‐selectively labeled NS4B can be readily obtained using cell‐free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies., Membrane proteins revealed: We show how proton‐detected 100 kHz magic‐angle‐spinning solid‐state NMR can be harnessed for investigation of eukaryotic membrane proteins in lipids. Direct NMR screening of lipid environment on sub‐milligram amounts of protein guide optimal sample preparation. Amino‐acid‐selectively labeled protein from cell‐free synthesis opens the way to structural studies.

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.

Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years., Viruses, 12 (10), ISSN:1999-4915

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.

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.

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.

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.