119 research outputs found

    Hepatitis E virus RNA replication polyprotein: taking structural biology seriously

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    International audienceHepatitis E virus (HEV) infects approximately 20 million individuals each year allaround the world, both in developing and industrialized countries. It leads to 40,000–70,000 deaths annually, especially in immunocompromised patients and pregnant women.Despite its recognized major public health issue status and zoonotic potential, no specifictreatment is available. Indeed, HEV life cycle characterization is hampered by the lackof efficient infectious cell culture systems or in vivo models. A better knowledge of HEVvirology is therefore needed. By providing descriptions of the three-dimensional structuresof viral proteins at atomic level, structural biology can be a powerful tool to understandviral replication and help develop specific antivirals. In this comment, we describe how bothexperimental and advanced computational structural biology help to decipher HEV virologyand make a case for heeding its lessons

    Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

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    Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition

    Structural biology. Kickstarting a viral RNA polymerase.

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    Etude structurale de protéines virales impliquées dans la réplication et régulation du cycle de multiplication d'un virus de plante

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    Le virus de la mosaïque jaune du navet (TYMV) est un excellent modèle pour la réplication des virus à ARN simple brin de polarité positive. Ce petit virus de plante code l ensemble desprotéines nécessaires à sa réplication sous forme d un précurseur polyprotéine (206K). Du NauC-terminus, celui-ci porte une activité méthyltransférase, protéase à cystéine (PRO),hélicase et ARN-polymérase ARN dépendante (POL). Comme tous les virus à ARN(+)connus, son complexe de réplication est étroitement lié à des membranes cellulaires. Dans lecas du TYMV, c est l enveloppe des chloroplastes qui est impliquée. Un des acteurs clés de laréplication est la polymérase qui permet la synthèse de nouveaux génomes. La régulation de son activité implique dans un premier temps son clivage de la 206K par PRO. Ainsi libérée,elle est recrutée aux chloroplastes par une interaction directe avec le domaine PRO du produit de clivage 140K, afin de former le complexe de réplication. Un second clivage par PRO contenu dans 140K, à la jonction PRO-hélicase, permet de poursuivre le cycle de réplication.Récemment, il a également été montré que l activité POL était régulée par la voie ubiquitine-protéasome durant le cycle viral. Ubiquitinilée par la cellule hôte, elle est adressée au protéasome où elle sera dégradée. Cependant, PRO, grâce à sa seconde fonction ubiquitine hydrolase, est capable de la protéger de cette dégradation. Afin de caractériser d un point de vue structural ce mécanisme de régulation de la réplication, nous avons cristallisé, à l aide d un contaminant, PRO et avons résolu sa structure. L empilement cristallin est tel que le Cterminus d un domaine PRO est inséré dans la crevasse catalytique du domaine PRO suivant,nous fournissant ainsi des informations structurales sur son activité endopeptidase. Dans un second temps, afin d avoir des informations sur sa seconde activité, nous avons réalisé un complexe stable entre PRO et une molécule d ubiquitine afin de le cristalliser et résoudre sa structure. Enfin, nous avons initié l étude cristallographique de la polymérase.Turnip Yellow Mosaic Virus is an excellent model for positive-stranded RNA virus replication. It s a small plant virus whose replication machinery is encoded in the viralgenome as a single polyprotein (206K). From N- to C-terminus, this 206K harbors amethyltransferase, a cysteine proteinase (PRO), a helicase and an RNA-dependent RNApolymerase (POL). As in all RNA(+) viruses known, the replication complex is bound to cellmembranes. For TYMV, it ,is the chloroplast envelope that is implicated. A key component inthe replication process is the polymerase, that allows the synthesis of new genomes. The regulation of its activity involves initially its cleavage by PRO from the 206K. Once liberated,the polymerase is recruited to the chloroplasts through a direct interaction with the PROdomain, contained in 140K, in order to form the replication complex. A second cleavage of140K by PRO at the PRO-helicase junction allows to continue the replication cycle. Recently,it has also been shown that the POL activity was regulated by the ubiquitin-proteasomesystem during the viral multiplication cycle. Ubiquitinilated by the host cell, POL is addressed to the proteasome where it is degraded. However, PRO, due to its second function as an ubiquitin hydrolase, is able to protect POL from its degradation. In order to characterizethis mechanism of replication regulation with a structural point of view, we crystallized,assisted by contaminant, PRO and resolved its structure. In the crystal packing, the C-terminalof a PRO domain is inserted into the catalytic cleft of the next PRO domain, thus providing us structural informations on its endopeptidase activity. In a second step, in order to obtain information about its second activity, we made a stable complex between PRO and amolecule of ubiquitin in order to cristallise it. Finally, we initiated the crystallographic studyof POL.Keywords:PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

    Crystallization of mutants of Turnip yellow mosaic virus protease/ubiquitin hydrolase designed to prevent protease self-recognition.

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    International audienceProcessing of the polyprotein of Turnip yellow mosaic virus is mediated by the protease PRO. PRO cleaves at two places, one of which is at the C-terminus of the PRO domain of another polyprotein molecule. In addition to this processing activity, PRO possesses an ubiquitin hydrolase (DUB) activity. The crystal structure of PRO has previously been reported in its polyprotein-processing mode with the C-terminus of one PRO inserted into the catalytic site of the next PRO, generating PRO polymers in the crystal packing of the trigonal space group. Here, two mutants designed to disrupt specific PRO-PRO interactions were generated, produced and purified. Crystalline plates were obtained by seeding and cross-seeding from initial `sea urchin'-like microcrystals of one mutant. The plates diffracted to beyond 2 Å resolution at a synchrotron source and complete data sets were collected for the two mutants. Data processing and analysis indicated that both mutant crystals belonged to the same monoclinic space group, with two molecules of PRO in the asymmetric unit

    Replicative/transcription enzymes of Flaviviridae

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    The family Flaviviridae comprises several major human pathogens including Hepatitis C virus (genus hepacivirus), yellow fever virus or dengue virus (genus flavivirus). The Flaviviridae genomes are made of a single stranded RNA segment that encodes seven non-structural proteins : NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 in the flaviviruses like dengue virus and p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B for Hepatitis C virus. These proteins are expressed during the intracellular part of the infectious cycle, where they participate in transcribing and replicating the viral genome in the context of a membrane-bound multi-protein complex named the replication complex. Several of these proteins are endowed with one or multiple enzymatic activities and represent important targets against which specific antiviral inhibitors have been developed, several of which are currently used for therapy. Here, we review our current understanding of the molecular basis for viral RNA transcription and replication, focusing on polymerases and protease-helicases

    Structure of the prefusion form of the vesicular stomatitis virus glycoprotein G.

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    International audienceGlycoprotein G of the vesicular stomatitis virus triggers membrane fusion via a low pH-induced structural rearrangement. Despite the equilibrium between the pre- and postfusion states, the structure of the prefusion form, determined to 3.0 angstrom resolution, shows that the fusogenic transition entails an extensive structural reorganization of G. Comparison with the structure of the postfusion form suggests a pathway for the conformational change. In the prefusion form, G has the shape of a tripod with the fusion loops exposed, which point toward the viral membrane, and with the antigenic sites located at the distal end of the molecule. A large number of G glycoproteins, perhaps organized as in the crystals, act cooperatively to induce membrane merging

    De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation

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    International audienceHepatitis E virus (HEV), a major cause of acute viral hepatitis, is a single-stranded, positive-sense RNA virus. As such, it encodes a 1700-residue replication polyprotein pORF1 that directs synthesis of new viral RNA in infected cells. Here we report extensive modeling with AlphaFold2 of the full-length pORF1, and its production by in vitro translation. From this, we give a detailed update on the breakdown into domains of HEV pORF1. We also provide evidence that pORF1’s N-terminal domain is likely to oligomerize to form a dodecameric pore, homologously to what has been described for Chikungunya virus. Beyond providing accurate folds for its five domains, our work highlights that there is no canonical protease encoded in pORF1 and that flexibility in several functionally important regions rather than proteolytic processing may serve to regulate HEV RNA synthesis

    Étude structurale de la protéine UL36 du HSV1

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    Session : Structure et morphogénèseNational audienc
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