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Publications Département Virologie


  • A. Alard, C. Marboeuf, B. Fabre, C. Jean, Y. Martineau, F. Lopez, P. Vende, D. Poncet, R. J. Schneider, C. Bousquet, et S. Pyronnet, « Differential Regulation of the Three Eukaryotic mRNA Translation Initiation Factor (eIF) 4Gs by the Proteasome », Frontiers in Genetics, vol. 10, p. 254, mars 2019.
    Résumé : The 4G family of eukaryotic mRNA translation initiation factors is composed of three members (eIF4GI, eIF4GII, and DAP5). Their specific roles in translation initiation are under intense investigations, but how their respective intracellular amounts are controlled remains poorly understood. Here we show that eIF4GI and eIF4GII exhibit much shorter half-lives than that of DAP5. Both eIF4GI and eIF4GII proteins, but not DAP5, contain computer-predicted PEST motifs in their N-termini conserved across the animal kingdom. They are both sensitive to degradation by the proteasome. Under normal conditions, eIF4GI and eIF4GII are protected from proteasomal destruction through binding to the detoxifying enzyme NQO1 [NAD(P)H:quinone oxidoreductase]. However, when cells are exposed to oxidative stress both eIF4GI and eIF4GII, but not DAP5, are degraded by the proteasome in an N-terminal-dependent manner, and cell viability is more compromised upon silencing of DAP5. These findings indicate that the three eIF4G proteins are differentially regulated by the proteasome and that persistent DAP5 plays a role in cell survival upon oxidative stress.
    Mots-clés : 20s proteasome, DAP5, NQO1, Nrf2, Oxidative Stress, Pest, Proteasome, ROTA, VIRO.
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  • L. Belot, A. Albertini, et Y. Gaudin, « Structural and cellular biology of rhabdovirus entry », Advances in Virus Research, vol. 104, p. 147-183, 2019.
    Résumé : Rhabdoviruses are enveloped viruses with a negative-sense single strand RNA genome and are widespread among a great variety of organisms. In their membrane, they have a single glycoprotein (G) that mediates both virus attachment to cellular receptors and fusion between viral and endosomal membranes allowing viral genome release in the cytoplasm. We present structural and cellular aspects of Rhabdovirus entry into their host cell with a focus on vesicular stomatitis virus (VSV) and rabies virus (RABV) for which the early events of the viral cycle have been extensively studied. Recent data have shown that the only VSV receptors are the members of the LDL-R family. This is in contrast with RABV for which multiple receptors belonging to unrelated families have been identified. Despite having different receptors, after attachment, rhabdovirus internalization occurs through clathrin-mediated endocytosis (CME) in an actin-dependent manner. There are still debates about the exact endocytic pathway of VSV in the cell and on RABV transport in the neuronal axon. In any case, fusion is triggered in the endosomal vesicle via a low-pH induced structural rearrangement of G from its pre- to its postfusion conformation. Vesiculovirus G is one of the best characterized fusion glycoproteins as the previously reported crystal structures of the pre- and postfusion states have been recently completed by those of intermediates during the structural transition. Understanding the entry pathway of rhabdoviruses may have strong impact in biotechnologies as, for example, VSV G is used for pseudotyping lentiviruses to promote efficient transduction, and VSV is a promising oncolytic virus.
    Mots-clés : Chandipura virus, Clathrin-mediated endocytosis, Glycoprotein, LDL-receptor, Membrane fusion, Rabies virus, RHABDO, Rhabdovirus, Vesicular stomatitis virus, Viral receptor, VIRO.

  • L. Belot, A. Albertini, et Y. Gaudin, « Chapter Five - Structural and cellular biology of rhabdovirus entry », in Advances in Virus Research, vol. 104, M. Kielian, T. C. Mettenleiter, et M. J. Roossinck, Éd. Academic Press, 2019, p. 147-183.
    Résumé : Rhabdoviruses are enveloped viruses with a negative-sense single strand RNA genome and are widespread among a great variety of organisms. In their membrane, they have a single glycoprotein (G) that mediates both virus attachment to cellular receptors and fusion between viral and endosomal membranes allowing viral genome release in the cytoplasm. We present structural and cellular aspects of Rhabdovirus entry into their host cell with a focus on vesicular stomatitis virus (VSV) and rabies virus (RABV) for which the early events of the viral cycle have been extensively studied. Recent data have shown that the only VSV receptors are the members of the LDL-R family. This is in contrast with RABV for which multiple receptors belonging to unrelated families have been identified. Despite having different receptors, after attachment, rhabdovirus internalization occurs through clathrin-mediated endocytosis (CME) in an actin-dependent manner. There are still debates about the exact endocytic pathway of VSV in the cell and on RABV transport in the neuronal axon. In any case, fusion is triggered in the endosomal vesicle via a low-pH induced structural rearrangement of G from its pre- to its postfusion conformation. Vesiculovirus G is one of the best characterized fusion glycoproteins as the previously reported crystal structures of the pre- and postfusion states have been recently completed by those of intermediates during the structural transition. Understanding the entry pathway of rhabdoviruses may have strong impact in biotechnologies as, for example, VSV G is used for pseudotyping lentiviruses to promote efficient transduction, and VSV is a promising oncolytic virus.
    Mots-clés : Chandipura virus, Clathrin-mediated endocytosis, Glycoprotein, LDL-receptor, Membrane fusion, Rabies virus, RHABDO, Rhabdovirus, Vesicular stomatitis virus, Viral receptor, VIRO.

  • M. S. Deffieu, T. D. Alayi, C. Slomianny, et S. Tomavo, « The Toxoplasma gondii dense granule protein TgGRA3 interacts with host Golgi and dysregulates anterograde transport », Biology Open, vol. 8, nᵒ 3, p. UNSP bio039818, mars 2019.
    Résumé : After entry into the host cell, the intracellular parasite Toxoplasma gondii resides within a membrane-bound compartment, the parasitophorous vacuole (PV). The PV defines an intracellular, parasite-specific niche surrounded by host organelles, including the Golgi apparatus. The mechanism by which T. gondii hijacks the host Golgi and subverts its functions remains unknown. Here, we present evidence that the dense granule protein TgGRA3 interacts with host Golgi, leading to the formation of tubules and the entry of host Golgi material into the PV. Targeted disruption of the TgGRA3 gene delays this engulfment of host Golgi. We also demonstrate that TgGRA3 oligomerizes and binds directly to host Golgi membranes. In addition, we show that TgGRA3 dysregulates anterograde transport in the host cell, thereby revealing one of the mechanisms employed by T. gondii to recruit host organelles and divert their functions. This article has an associated First Person interview with the first author of the paper.
    Mots-clés : Anterograde transport, Anterograde transport, association, BMCT, cell endoplasmic-reticulum, cholesterol, Dense granules, domains, gra3, Host Golgi, invasion, nanotubular network, parasitophorous vacuole membrane, Secretion, Toxoplasma gondii, VIRO.

  • M. Duarte, P. Vende, A. Charpilienne, M. Gratia, C. Laroche, et D. Poncet, « Rotavirus Infection Alters Splicing of the Stress-Related Transcription Factor XBP1 », Journal of Virology, vol. 93, nᵒ 5, p. e01739-18, mars 2019.
    Résumé : XBP1 is a stress-regulated transcription factor also involved in mammalian host defenses and innate immune response. Our investigation of XBP1 RNA splicing during rotavirus infection revealed that an additional XBP1 RNA ( XBP1es) that corresponded to exon skipping in the XBP1 pre-RNA is induced depending on the rotavirus strain used. We show that the translation product of XBP1es (XBP1es) has trans-activation properties similar to those of XBP1 on ER stress response element (ERSE) containing promoters. Using monoreassortant between ES+ ("skipping") and ES-("nonskipping") strains of rotavirus, we show that gene 7 encoding the viral translation enhancer NSP3 is involved in this phenomenon and that exon skipping parallels the nuclear relocalization of cytoplasmic PABP. We further show, using recombinant rotaviruses carrying chimeric gene 7, that the ES+ phenotype is linked to the eIF4G-binding domain of NSP3. Because the XBP1 transcription factor is involved in stress and immunological responses, our results suggest an alternative way to activate XBP1 upon viral infection or nuclear localization of PABP. IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. Here we show that infection with several rotavirus strains induces an alternative splicing of the RNA encoding the stressed-induced transcription factor XBP1. The genetic determinant of XBP1 splicing is the viral RNA translation enhancer NSP3. Since XBP1 is involved in cellular stress and immune responses and since the XBP1 protein made from the alternatively spliced RNA is an active transcription factor, our observations raise the question of whether alternative splicing is a cellular response to rotavirus infection.
    Mots-clés : consensus sequence, endoplasmic-reticulum stress, ifn-beta induction, immune response, messenger-rna, nsp3, nuclear import, nuclear transport, nucleocytoplasmic transport, pabp, poly(a) binding-protein, poly(a)-binding protein, reverse genetics, ROTA, rotavirus, splicing, stress response, unfolded protein response, VIRO, xbp1.

  • A. Huet, R. L. Duda, P. Boulanger, et J. F. Conway, « Capsid expansion of bacteriophage T5 revealed by high resolution cryoelectron microscopy », Proceedings of the National Academy of Sciences of the United States of America, oct. 2019.
    Résumé : The large (90-nm) icosahedral capsid of bacteriophage T5 is composed of 775 copies of the major capsid protein (mcp) together with portal, protease, and decoration proteins. Its assembly is a regulated process that involves several intermediates, including a thick-walled round precursor prohead that expands as the viral DNA is packaged to yield a thin-walled and angular mature capsid. We investigated capsid maturation by comparing cryoelectron microscopy (cryo-EM) structures of the prohead, the empty expanded capsid both with and without decoration protein, and the virion capsid at a resolution of 3.8 Å for the latter. We detail the molecular structure of the mcp, its complex pattern of interactions, and their evolution during maturation. The bacteriophage T5 mcp is a variant of the canonical HK97-fold with a high level of plasticity that allows for the precise assembly of a giant macromolecule and the adaptability needed to interact with other proteins and the packaged DNA.
    Mots-clés : bacteriophage, capsid, conformational change, cryoelectron microscopy, T5PHAG, VIRO.

  • Z. Lama, Y. Gaudin, D. Blondel, et C. Lagaudriere-Gesbert, « Kinase inhibitors Tyrphostin 9 and rottlerin block early steps of rabies virus cycle », Antiviral Research, vol. 168, p. 51-60, août 2019.
    Résumé : Rabies virus (RABV) is a neurotropic virus that causes fatal encephalitis in humans and animals and still kills up to 59,000 people worldwide every year. To date, only preventive or post-exposure vaccination protects against the disease but therapeutics are missing. After screening a library of 80 kinases inhibitors, we identified two compounds as potent inhibitors of RABV infection: Tyrphostin 9 and Rottlerin. Mechanism of action studies show that both inhibitors interfere with an early step of viral cycle and can prevent viral replication. In presence of Tyrphostin 9, the viral entry through endocytosis is disturbed leading to improper delivery of viral particles in cytoplasm, whereas Rottlerin is inhibiting the transcription, most likely by decreasing intracellular ATP concentrations, and therefore the replication of the viral genome.
    Mots-clés : Entry inhibitor, Rabies virus (RABV), RHABDO, Rottlerin, Transcription inhibitor, Tyrphostin 9, VIRO.

  • A. Moris, M. Pereira, et L. Chakrabarti, « A role for antibodies in natural HIV control », Current opinion in HIV and AIDS, vol. 14, nᵒ 4, p. 265-272, juill. 2019.
    Résumé : PURPOSE OF REVIEW: Rare patients naturally control HIV replication without antiretroviral therapy. Understanding the mechanisms implicated in natural HIV control will inform the development of immunotherapies against HIV. Elite controllers are known for developing efficient antiviral T-cell responses, but recent findings suggest that antibody responses also play a significant role in HIV control. We review the key studies that uncovered a potent memory B-cell response and highly functional anti-HIV antibodies in elite controllers, and explore the mechanisms that may account for the distinct properties of their humoral response. RECENT FINDINGS: Elite controllers maintain a large HIV-specific memory B-cell pool that is sustained by efficient T follicular helper function. Neutralizing antibody rarely show high titers in controllers, but seem capable, at least in certain cases, of neutralizing contemporaneous viral strains. In addition, elite controllers display a unique HIV-specific antibody profile in terms of isotype, antigen specificity, and glycosylation pattern, resulting in polyfunctional antibody effector functions that may promote infected cell lysis and prime effectors of the antiviral immune response. SUMMARY: Lessons from elite controller studies argue for the importance of integrating the many parameters defining a polyfunctional antibody response when evaluating candidate vaccines and immunotherapeutic approaches directed at HIV.
    Mots-clés : HERPES, VIRO.

  • J. Nikolic, C. Lagaudrière-Gesbert, N. Scrima, D. Blondel, et Y. Gaudin, « Structure and Function of Negri Bodies », Advances in Experimental Medicine and Biology, vol. 1140, p. 111-127, 2019.
    Résumé : Replication and assembly of many viruses occur in viral factories which are specialized intracellular compartments formed during viral infection. For rabies virus, those viral factories are called Negri bodies (NBs). NBs are cytoplasmic inclusion bodies in which viral RNAs (mRNAs as well as genomic and antigenomic RNAs) are synthesized. NBs are spherical, they can fuse together, and can reversibly deform when encountering a physical barrier. All these characteristics are similar to those of eukaryotic membrane-less liquid organelles which contribute to the compartmentalization of the cell interior. Indeed, the liquid nature of NBs has been confirmed by FRAP experiments. The co-expression of rabies virus nucleoprotein N and phosphoprotein P is sufficient to induce the formation of cytoplasmic inclusions recapitulating NBs properties. Remarkably, P and N have features similar to those of cellular proteins involved in liquid organelles formation: N is an RNA-binding protein and P contains intrinsically disordered domains. An overview of the literature indicates that formation of liquid viral factories by phase separation is probably common among Mononegavirales. This allows specific recruitment and concentration of viral proteins. Finally, as virus-associated molecular patterns recognized by cellular sensors of RNA virus replication are probably essentially present in the viral factory, there should be a subtle interplay (which remains to be characterized) between those liquid structures and the cellular proteins which trigger the innate immune response.
    Mots-clés : Liquid organelles, Negri bodies, Phase separation, Rabies virus, RHABDO, Viral factory, VIRO.

  • N. Poccardi, A. Rousseau, O. Haigh, J. Takissian, T. Naas, C. Deback, L. Trouillaud, M. Issa, S. Roubille, F. Juillard, S. Efstathiou, P. Lomonte, et M. Labetoulle, « HSV-1 replication, ocular disease and reactivations from latency are restricted unilaterally after inoculation of virus into the lip », Journal of Virology, sept. 2019.
    Résumé : Ocular herpes simplex keratitis (HSK) is a consequence of viral reactivations from trigeminal ganglia (TG) and occurs almost exclusively in the same eye in humans. In our murine oro-ocular (OO) model, HSV-1 inoculation in one side of the lip propagates virus to infect the ipsilateral TG. Replication here allows infection of the brainstem, and infection of the contralateral TG. Interestingly, HSK is observed in our OO model only from the eye ipsilateral to site of lip infection. Thus, unilateral restriction of HSV-1 may be due to differential kinetics of virus arrival in the ipsilateral versus contralateral TG. We inoculated mice with HSV-1 reporter viruses, and then superinfected them to monitor changes in acute and latent phase gene expression in TG after superinfection, compared to control (single inoculation). Delaying superinfection by 4 days after initial right lip inoculation elicited failed superinfecting-virus gene expression, and eliminated clinical signs of disease. Initial inoculation with a thymidine kinase-deficient HSV-1 (TKdel) completely abolished reactivation of WT superinfecting virus from TG during latent stage. In light of these seemingly failed infections, viral genome was detected in both TGs. Our data demonstrate that inoculation of HSV-1 in the lip propagates virus to both TG, but with delay in reaching the TG contralateral to the side of lip infection. This delay is responsible for restricting viral replication to the ipsilateral TG, which abrogates ocular disease and viral reactivations from the contralateral side. These observations may help to understand why HSK is observed unilaterally in humans, and provides insight into vaccine strategies to protect against HSK.IMPORTANCEHerpetic keratitis (HK) is the leading cause of blindness by an infectious agent in the developed world. This disease can occur after reactivation of Herpes simplex virus 1 in the trigeminal ganglia leading to dissemination of virus to, and infection of, the cornea. A clinical paradox is evidenced by the bilateral presence of latent viral genomes in both trigeminal ganglia, while for any given patient the disease is unilateral with recurrences in a single eye. Our study links the kinetics of early infection to unilateral disease phenomenon, and demonstrates protection against viral reactivation when kinetics are exploited. Our results have direct implications in the understanding of human disease pathogenesis, and immunotherapeutic strategies for the treatment of HK and viral reactivations.
    Mots-clés : HERPES, VIRO.

  • M. Schönfeld, U. Knackmuss, P. Chandorkar, P. Hörtnagl, T. J. Hope, A. Moris, R. Bellmann-Weiler, C. Lass-Flörl, W. Posch, et D. Wilflingseder, « Co- but not Sequential Infection of DCs Boosts Their HIV-Specific CTL-Stimulatory Capacity », Frontiers in Immunology, vol. 10, p. 1123, 2019.
    Résumé : Pathogenic bacteria and their microbial products activate dendritic cells (DCs) at mucosal surfaces during sexually transmitted infections (STIs) and therefore might also differently shape DC functions during co-infection with HIV-1. We recently illustrated that complement (C) coating of HIV-1 (HIV-C), as primarily found during the acute phase of infection before appearance of HIV-specific antibodies, by-passed SAMHD1-mediated restriction in DCs and therefore mediated an increased DC activation and antiviral capacity. To determine whether the superior antiviral effects of HIV-C-exposed DCs also apply during STIs, we developed a co-infection model in which DCs were infected with Chlamydia spp. simultaneously (HIV-C/Chlam-DCs or HIV/Chlam-DCs) or a sequential infection model, where DCs were exposed to Chlamydia for 3 or 24 h (Chlam-DCs) followed by HIV-1 infection. Co-infection of DCs with HIV-1 and Chlamydia significantly boosted the CTL-stimulatory capacity compared to HIV-1-loaded iDCs and this boost was independent on the opsonization pattern. This effect was lost in the sequential infection model, when opsonized HIV-1 was added delayed to Chlamydia-loaded DCs. The reduction in the CTL-stimulatory capacity of Chlam-DCs was not due to lower HIV-1 binding or infection compared to iDCs or HIV-C/Chlam-DCs, but due to altered fusion and internalization mechanisms within DCs. The CTL-stimulatory capacity of HIV-C in Chlam-DCs correlated with significantly reduced viral fusion compared to iDCs and HIV-C/Chlam-DCs and illustrated considerably increased numbers of HIV-C-containing vacuoles than iDCs. The data indicate that Chlamydia co-infection of DCs mediates a transient boost of their HIV-specific CTL-stimulatory and antiviral capacity, while in the sequential infection model this is reversed and associated with hazard to the host.
    Mots-clés : activation, capture, complement, CTL, HERPES, HIV-1, STIs, VIRO.

  • C. Taisne, M. Lussignol, E. Hernandez, A. Moris, L. Mouna, et A. Esclatine, « Human cytomegalovirus hijacks the autophagic machinery and LC3 homologs in order to optimize cytoplasmic envelopment of mature infectious particles », Scientific Reports, vol. 9, nᵒ 1, p. 4560, mars 2019.
    Résumé : During its life cycle, Human cytomegalovirus (HCMV) tightly modulates autophagy, a vesicular pathway allowing degradation and recycling of cellular components. To study the interplay between autophagy and the viral life cycle, we established various autophagy-deficient human fibroblastic cell lines. By knocking down the expression or activity of five autophagy-related proteins, we confirmed the proviral function that the autophagic machinery exerts on HCMV production. Using 3D reconstruction from confocal microscopy and electron microscopy, we demonstrated that lipidated LC3-positive vesicles accumulated at the viral assembly compartment (vAC). The vAC is a juxtanuclear ring-shaped structure containing several organelles and membranes, where assembly and final envelopment of HCMV particles occur. Two LC3 homologs, GABARAPL1 and GATE16, also accumulated during HCMV infection and were associated with the vAC, in proximity with fragmented Golgi stacks. Additionally, we observed the formation of a pre-assembly compartment (PrAC) in infected cells, which consists of a juxtanuclear structure containing both fragmented Golgi and LC3-positive vesicles. Finally, we showed that highly purified extracellular viral particles were associated with various autophagy proteins. Our results thus suggest that autophagy machinery participates to the final cytoplasmic envelopment of HCMV viral particles into the vAC and that autophagy-related proteins can be spotted in the virions.
    Mots-clés : blocks, cells, compartment, complex, entry, HERPES, inhibition, protein, time, trs1, VIRO, virus.

  • V. U. Weiss, R. Pogan, S. Zoratto, K. M. Bond, P. Boulanger, M. F. Jarrold, N. Lyktey, D. Pahl, N. Puffler, M. Schelhaas, E. Selivanovitch, C. Uetrecht, et G. Allmaier, « Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA) », Analytical and Bioanalytical Chemistry, vol. 411, nᵒ 23, p. 5951-5962, sept. 2019.
    Résumé : (Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation. Graphical abstract.
    Mots-clés : DMA, Mass spectrometry, Molecular weight/mass, Native nES GEMMA, Size, T5PHAG, VIRO, VLP.


  • A. Abou-Hamdan, L. Belot, A. Albertini, et Y. Gaudin, « Monomeric Intermediates Formed by Vesiculovirus Glycoprotein during Its Low-pH-induced Structural Transition », Journal of Molecular Biology, avr. 2018.
    Mots-clés : conformational change, glycoprotein, Influenza, membrane fusion, RHABDO, Vesiculovirus, VIRO.

  • M. Ansaldi, L. Debarbieux, S. Gandon, M. - A. Petit, P. Tavares, et P. Boulanger, « "French Phage Network"-Third Meeting Report », Viruses, vol. 10, nᵒ 3, mars 2018.
    Résumé : In its third year of existence, the French Phage Network ( is pursuing its expansion. With more than 25 groups, mostly based in France, working on the various aspects of phage research, the network has increased its visibility, interactivity, and activity. The third meeting of the network, held on November 2017 at the Gif-sur-Yvette Centre National de la Recherche Scientifique (CNRS) campus, was a great opportunity for many young scientists to present their work and interact with more senior scientists, amongst which several were invited from abroad. Here we provide a summary of the work presented at this occasion during the oral presentations and poster sessions.
    Mots-clés : bacteria, bacteriophage, co-evolution, France, genomics, infection, PHAG+, phage therapy, resistance, structural biology, T5PHAG, VIRO, virulence.

  • C. Cohen, A. Corpet, S. Roubille, M. A. Maroui, N. Poccardi, A. Rousseau, C. Kleijwegt, O. Binda, P. Texier, N. Sawtell, M. Labetoulle, et P. Lomonte, « Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis », PLoS pathogens, vol. 14, nᵒ 9, p. e1007313, sept. 2018.
    Résumé : Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact contribution is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML NBs, leading to the formation of viral DNA-containing PML NBs (vDCP NBs), and the complete silencing of HSV-1. Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP and Re-ChIP show that vDCP NBs-associated latent/quiescent viral genomes are chromatinized almost exclusively with H3.3 modified on its lysine (K) 9 by trimethylation, consistent with an interaction of the H3.3 chaperones with multiple viral loci and with the transcriptional silencing of HSV-1. Only simultaneous inactivation of both H3.3 chaperone complexes has a significant impact on the deposition of H3.3 on viral genomes, suggesting a compensation mechanism. In contrast, the sole depletion of PML significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. vDCP NBs-associated HSV-1 genomes are not definitively silenced since the destabilization of vDCP NBs by ICP0, which is essential for HSV-1 reactivation in vivo, allows the recovery of a transcriptional lytic program and the replication of viral genomes. Consequently, the present study demonstrates a specific chromatin regulation of vDCP NBs-associated latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML NB/histone H3.3/H3.3 chaperone axis.
    Mots-clés : HERPES, VIRO.

  • S. Dominguez-Medina, S. Fostner, M. Defoort, M. Sansa, A. - K. Stark, M. A. Halim, E. Vernhes, M. Gely, G. Jourdan, T. Alava, P. Boulanger, C. Masselon, et S. Hentz, « Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators », Science (New York, N.Y.), vol. 362, nᵒ 6417, p. 918-922, nov. 2018.
    Résumé : Measurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100.
    Mots-clés : bacteriophage-t5, resolution, T5PHAG, VIRO.

  • F. El Asmi, C. E. Brantis-de-Carvalho, D. Blondel, et M. K. Chelbi-Alix, « Rhabdoviruses, Antiviral Defense, and SUMO Pathway », Viruses-Basel, vol. 10, nᵒ 12, p. 686, déc. 2018.
    Résumé : Small Ubiquitin-like MOdifier (SUMO) conjugation to proteins has essential roles in several processes including localization, stability, and function of several players implicated in intrinsic and innate immunity. In human, five paralogs of SUMO are known of which three are ubiquitously expressed (SUMO1, 2, and 3). Infection by rhabdoviruses triggers cellular responses through the activation of pattern recognition receptors, which leads to the production and secretion of interferon. This review will focus on the effects of the stable expression of the different SUMO paralogs or Ubc9 depletion on rhabdoviruses-induced interferon production and interferon signaling pathways as well as on the expression and functions of restriction factors conferring the resistance to rhabdoviruses.
    Mots-clés : host sumoylation, IFN, MxA, PKR, RHABDO, SUMO, VIRO.

  • L. M. Godinho, M. E. S. Fadel, C. Monniot, L. Jakutyte, I. Auzat, A. Labarde, K. Djacem, L. Oliveira, R. Carballido-Lopez, S. Ayora, et P. Tavares, « The Revisited Genome of Bacillus subtilis Bacteriophage SPP1 », Viruses-Basel, vol. 10, nᵒ 12, p. 705, déc. 2018.
    Résumé : Bacillus subtilis bacteriophage SPP1 is a lytic siphovirus first described 50 years ago. Its complete DNA sequence was reported in 1997. Here we present an updated annotation of the 44,016 bp SPP1 genome and its correlation to different steps of the viral multiplication process. Five early polycistronic transcriptional units encode phage DNA replication proteins and lysis functions together with less characterized, mostly non-essential, functions. Late transcription drives synthesis of proteins necessary for SPP1 viral particles assembly and for cell lysis, together with a short set of proteins of unknown function. The extensive genetic, biochemical and structural biology studies on the molecular mechanisms of SPP1 DNA replication and phage particle assembly rendered it a model system for tailed phages research. We propose SPP1 as the reference species for a new SPP1-like viruses genus of the Siphoviridae family.
    Mots-clés : Bacillus subtilis, Bacteriophage, BIM, complete nucleotide-sequence, crystal-structure, DBG, DNA replication, dna-replication, functional-analysis, genome organization, nucleoprotein complex, PHAG+, phage spp1, portal protein, Siphoviridae, small-subunit, spp1, structural rearrangements, to-tail interface, VIRO, virus assembly, virus DNA packaging, virus evolution.

  • M. Hermouet, M. Sansa, M. Defoort, L. Banniard, S. Dominguez-Medina, S. Fostner, U. Palanchoke, A. Fafin, M. Gely, L. Hutin, C. Plantier, E. Rolland, C. Tabone, G. Usai, T. Ernst, P. Villard, G. Billiot, P. Mattei, G. Nonglaton, C. Fontelaye, C. Barrois, O. Castany, E. G. Santos, P. E. Allain, E. Vernhes, P. Boulanger, A. Brenac, C. Masselon, I. Favero, T. Alava, G. Jourdan, et S. Hentz, « Very Large Scale Integration Optomechanics: a cure for loneliness of NEMS resonators? », in 2018 Ieee International Electron Devices Meeting (iedm), New York: Ieee, 2018.
    Résumé : The first Very Large Scale Integration process with variable shape beam lithography for optomechanical devices is presented. State of the art performance was obtained with silicon microdisk resonators showing 1 million optical quality factors and 10(-17) m.Hz((-1/2)) displacement resolution. Single-particle mass spectrometry could be performed with these optomechanical resonators in vacuum. The devices retained high performance when directly immersed in liquid media, allowing for biosensing experiments. These results open the door to large, dense arrays of optomechanical sensors.
    Mots-clés : T5PHAG, VIRO.

  • J. Nikolic, L. Belot, H. Raux, P. Legrand, Y. Gaudin, et A. A. Albertini, « Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein », Nature Communications, vol. 9, nᵒ 1, p. 1029, 2018.

  • J. Nikolic, C. Lagaudrière-Gesbert, N. Scrima, D. Blondel, et Y. Gaudin, « [Rabies virus factories are formed by liquid-liquid phase separation] », Medecine Sciences: M/S, vol. 34, nᵒ 3, p. 203-205, mars 2018.

  • P. Tavares, « The Bacteriophage Head-to-Tail Interface », Sub-Cellular Biochemistry, vol. 88, p. 305-328, 2018.
    Résumé : Many icosahedral viruses use a specialized portal vertex for genome encapsidation in the viral capsid (or head). This structure then controls release of the viral genetic information to the host cell at the beginning of infection. In tailed bacteriophages, the portal system is connected to a tail device that delivers their genome to the bacterial cytoplasm. The head-to-tail interface is a multiprotein complex that locks the viral DNA inside the phage capsid correctly positioned for egress and that controls its ejection when the viral particle interacts with the host cell receptor. Here we review the molecular mechanisms how this interface is assembled and how it carries out those two critical steps in the life cycle of tailed phages.
    Mots-clés : Bacteriophage, Capsid, DNA ejection, DNA packaging, Head-to-tail interface, PHAG+, Portal system, VIRO.


  • C. - A. Arnaud, G. Effantin, C. Vivès, S. Engilberge, M. Bacia, P. Boulanger, E. Girard, G. Schoehn, et C. Breyton, « Bacteriophage T5 tail tube structure suggests a trigger mechanism for Siphoviridae DNA ejection », Nature Communications, vol. 8, nᵒ 1, p. 1953, déc. 2017.
    Résumé : The vast majority of phages, bacterial viruses, possess a tail ensuring host recognition, cell wall perforation and safe viral DNA transfer from the capsid to the host cytoplasm. Long flexible tails are formed from the tail tube protein (TTP) polymerised as hexameric rings around and stacked along the tape measure protein (TMP). Here, we report the crystal structure of T5 TTP pb6 at 2.2 Å resolution. Pb6 is unusual in forming a trimeric ring, although structure analysis reveals homology with all classical TTPs and related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin). Structures of T5 tail tubes before and after interaction with the host receptor were determined by cryo-electron microscopy at 6 Å resolution. Comparison of these two structures reveals that host-binding information is not propagated to the capsid through conformational changes in the tail tube, suggesting a role of the TMP in this information transduction process.
    Mots-clés : T5PHAG, VIRO.

  • E. Baquero, A. A. Albertini, H. Raux, A. Abou‐Hamdan, E. Boeri‐Erba, M. Ouldali, L. Buonocore, J. K. Rose, J. Lepault, S. Bressanelli, et Y. Gaudin, « Structural intermediates in the fusion‐associated transition of vesiculovirus glycoprotein », The EMBO Journal, vol. 36, nᵒ 5, p. 679-692, mars 2017.
    Mots-clés : B3S, conformational change, CRYOEM, glycoprotein, IMAPP, intermediate structures, membrane fusion, PF, RHABDO, Vesiculovirus, VIRO, VIROEM.

  • P. Cuniasse, P. Tavares, E. V. Orlova, et S. Zinn-Justin, « Structures of biomolecular complexes by combination of NMR and cryoEM methods », Current Opinion in Structural Biology, vol. 43, p. 104-113, 2017.

  • K. Djacem, P. Tavares, et L. Oliveira, « Bacteriophage SPP1 pac Cleavage: A Precise Cut without Sequence Specificity Requirement », Journal of Molecular Biology, janv. 2017.
    Résumé : In many tailed bacteriophages, DNA packaging is initiated by recognition and cleavage of a specific sequence pac by the small (TerS) and large (TerL) terminase subunits. It was previously shown that the SPP1 pac region has two sequences where TerS binds (pacR and pacL), flanking the segment where TerL cleaves the SPP1 DNA (pacC). However, the pac-specific sequences required to achieve this endonucleolytic cut were not established. Their characterization is essential to understand the underlying mechanism. We show that the pacR sequence localized within 35bp downstream of the pac cut can be extensively degenerated, including its c1 and c2 repeats, and that only a disruption of a 5-bp polyadenine tract impairs the pac cleavage. This result together with deletion analysis of pacL shows that the specific DNA sequences required for targeting the terminase for pac cleavage are considerably shorter than the large region bound by TerS. Furthermore, extensive degeneration of the 6-bp target sequence within pacC where pac cleavage occurs reveals that TerL maintains, remarkably, its precise position of cleavage. Studies with SPP1-related phages show the conservation of the cut position, irrespective of the sequence variation in pacC and in pacR or the changes in pacL-pacC distance. Mechanistically, our data are compatible with a model in which TerS interactions with part of the pacL sequence and a poly-A tract in pacR are sufficient to orient very accurately the TerL nuclease to a defined pacC position. They also demonstrate that the resulting precise cut at pacC is independent of the targeted DNA sequence.
    Mots-clés : Bacteriophage, DNA packaging, genome recognition, pac cleavage, PHAG+, terminase, VIRO.

  • M. Lussignol et A. Esclatine, « Herpesvirus and Autophagy: “All Right, Everybody Be Cool, This Is a Robbery!” », Viruses, vol. 9, nᵒ 12, p. 372, déc. 2017.
    Mots-clés : amphisome, autophagosomes, autophagy, egress, envelopment, HERPES, herpesvirus, innate immunity, latency, reactivation, subversion, VIRO.

  • J. Nikolic et D. Blondel, « [Rabies virus induces the formation of stress granules, which are in close proximity to viral factories] », Medecine Sciences: M/S, vol. 33, nᵒ 11, p. 921-923, nov. 2017.

  • J. Nikolic, R. Le Bars, Z. Lama, N. Scrima, C. Lagaudrière-Gesbert, Y. Gaudin, et D. Blondel, « Negri bodies are viral factories with properties of liquid organelles », Nature Communications, vol. 8, nᵒ 1, p. 58, juill. 2017.
    Résumé : Replication of Mononegavirales occurs in viral factories which form inclusions in the host-cell cytoplasm. For rabies virus, those inclusions are called Negri bodies (NBs). We report that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase is confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Coexpression of rabies virus N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for Negri bodies-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response.Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.
    Mots-clés : IMAGIF, PF, PHOT, RHABDO, VIRO.

  • M. Ouldali, V. Maury, G. Nicolas, et J. Lepault, « Photosome membranes merge and organize tending towards rhombohedral symmetry when light is emitted », Journal of Structural Biology, déc. 2017.
    Résumé : Polynoid worm elytra emit light when mechanically or electrically stimulated. Specialized cells, the photocytes, contain light emitting machineries, the photosomes. Successive stimulations induce light intensity variations and show a coupling within and between photosomes. Here, we describe, using electron tomography of cryo-substituted elytra and freeze-fracturing, the structural transition associated to light emission: undulating tubules come closer, organize and their number forming photosomes increases. Two repeating undulating tubules in opposite phase compose the photosome. Undulations are located on three hexagonal layers that regularly repeat and are equally displaced, in x y and z. The tubule membranes within layers merge giving rise to rings that tend to obey to quasi-rhombohedral symmetry. Merging may result either from close-association, hemifusion (one leaflet fusion) or from fusion (two leaflets fusion). Although the resolution of tomograms is not sufficient to distinguish these three cases, freeze-fracturing shows that hemifusion is a frequent process that leads to an reversible anastomosed membrane complex favoring communications, appearing as a major coupling factor of photosome light emission.
    Mots-clés : Cell communication, Cryo-substitution, CRYOEM, Electron tomography, Fusion, Hemifusion, Membrane structure, PF, Photosome, VIRO, VIROEM.

  • I. Vergne, F. Lafont, L. Espert, A. Esclatine, et M. Biard-Piechaczyk, « Autophagie, protéines ATG et maladies infectieuses », médecine/sciences, vol. 33, nᵒ 3, p. 312-318, 2017.

  • E. Vernhes, M. Renouard, B. Gilquin, P. Cuniasse, D. Durand, P. England, S. Hoos, A. Huet, J. F. Conway, A. Glukhov, V. Ksenzenko, E. Jacquet, N. Nhiri, S. Zinn-Justin, et P. Boulanger, « Erratum: High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid », Scientific Reports, vol. 7, p. 43977, avr. 2017.

  • E. Vernhes, M. Renouard, B. Gilquin, P. Cuniasse, D. Durand, P. England, S. Hoos, A. Huet, J. F. Conway, A. Glukhov, V. Ksenzenko, E. Jacquet, N. Nhiri, S. Zinn-Justin, et P. Boulanger, « High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid », Scientific Reports, vol. 7, p. 41662, févr. 2017.


  • A. Brice, D. R. Whelan, N. Ito, K. Shimizu, L. Wiltzer-Bach, C. Y. Lo, D. Blondel, D. A. Jans, T. D. M. Bell, et G. W. Moseley, « Quantitative Analysis of the Microtubule Interaction of Rabies Virus P3 Protein: Roles in Immune Evasion and Pathogenesis », Scientific Reports, vol. 6, p. 33493, sept. 2016.
    Résumé : Although microtubules (MTs) are known to have important roles in intracellular transport of many viruses, a number of reports suggest that specific viral MT-associated proteins (MAPs) target MTs to subvert distinct MT-dependent cellular processes. The precise functional importance of these interactions and their roles in pathogenesis, however, remain largely unresolved. To assess the association with disease of the rabies virus (RABV) MAP, P3, we quantitatively compared the phenotypes of P3 from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derivative strain, Ni-CE. Using confocal/live-cell imaging and dSTORM super-resolution microscopy to quantify protein interactions with the MT network and with individual MT filaments, we found that the interaction by Ni-CE-P3 is significantly impaired compared with Ni-P3. This correlated with an impaired capacity to effect association of the transcription factor STAT1 with MTs and to antagonize interferon (IFN)/STAT1-dependent antiviral signaling. Importantly, we identified a single mutation in Ni-CE-P3 that is sufficient to inhibit MT-association and IFN-antagonist function of Ni-P3, and showed that this mutation alone attenuates the pathogenicity of RABV. These data provide evidence that the viral protein-MT interface has important roles in pathogenesis, suggesting that this interface could provide targets for vaccine/antiviral drug development.
    Mots-clés : RHABDO, VIRO.

  • S. Fernandes, A. Labarde, C. Baptista, L. Jakutytè, P. Tavares, et C. São-José, « A non-invasive method for studying viral DNA delivery to bacteria reveals key requirements for phage SPP1 DNA entry in Bacillus subtilis cells », Virology, vol. 495, p. 79-91, août 2016.
    Résumé : Bacteriophages use most frequently a tail apparatus to create a channel across the entire bacterial cell envelope to transfer the viral genome to the host cell cytoplasm, initiating infection. Characterization of this critical step remains a major challenge due to the difficulty to monitor DNA entry in the bacterium and its requirements. In this work we developed a new method to study phage DNA entry that has the potential to be extended to many tailed phages. Its application to study genome delivery of bacteriophage SPP1 into Bacillus subtilis disclosed a key role of the host cell membrane potential in the DNA entry process. An energized B. subtilis membrane and a millimolar concentration of calcium ions are shown to be major requirements for SPP1 DNA entry following the irreversible binding of phage particles to the receptor YueB.
    Mots-clés : B. subtilis, Bacteriophage, Calcium, Membrane potential, PHAG+, SPP1, VIRO, Virus entry, YueB.

  • M. de Frutos, A. Leforestier, J. Degrouard, N. Zambrano, F. Wien, P. Boulanger, S. Brasilès, M. Renouard, D. Durand, et F. Livolant, « Can Changes in Temperature or Ionic Conditions Modify the DNA Organization in the Full Bacteriophage Capsid? », The Journal of Physical Chemistry. B, vol. 120, nᵒ 26, p. 5975-5986, juill. 2016.
    Résumé : We compared four bacteriophage species, T5, λ, T7, and Φ29, to explore the possibilities of DNA reorganization in the capsid where the chain is highly concentrated and confined. First, we did not detect any change in DNA organization as a function of temperature between 20 to 40 °C. Second, the presence of spermine (4+) induces a significant enlargement of the typical size of the hexagonal domains in all phages. We interpret these changes as a reorganization of DNA by slight movements of defects in the structure, triggered by a partial screening of repulsive interactions. We did not detect any signal characteristic of a long-range chiral organization of the encapsidated DNA in the presence and in the absence of spermine.
    Mots-clés : B3S, FAAM, T5PHAG, VIRO.

  • M. Gratia, P. Vende, A. Charpilienne, H. C. Baron, C. Laroche, E. Sarot, S. Pyronnet, M. Duarte, et D. Poncet, « Challenging the Roles of NSP3 and Untranslated Regions in Rotavirus mRNA Translation », PloS One, vol. 11, nᵒ 1, p. e0145998, 2016.
    Résumé : Rotavirus NSP3 is a translational surrogate of the PABP-poly(A) complex for rotavirus mRNAs. To further explore the effects of NSP3 and untranslated regions (UTRs) on rotavirus mRNAs translation, we used a quantitative in vivo assay with simultaneous cytoplasmic NSP3 expression (wild-type or deletion mutant) and electroporated rotavirus-like and standard synthetic mRNAs. This assay shows that the last four GACC nucleotides of viral mRNA are essential for efficient translation and that both the NSP3 eIF4G- and RNA-binding domains are required. We also show efficient translation of rotavirus-like mRNAs even with a 5'UTR as short as 5 nucleotides, while more than eleven nucleotides are required for the 3'UTR. Despite the weak requirement for a long 5'UTR, a good AUG environment remains a requirement for rotavirus mRNAs translation.
    Mots-clés : 3' Untranslated Regions, 5' Untranslated Regions, Animals, Base Sequence, Cell Line, Cricetinae, Mutagenesis, Site-Directed, Protein Biosynthesis, RNA, Messenger, ROTA, Sequence Homology, Nucleic Acid, Transcription, Genetic, Viral Nonstructural Proteins, VIRO.

  • A. Huet, R. L. Duda, R. W. Hendrix, P. Boulanger, et J. F. Conway, « Correct Assembly of the Bacteriophage T5 Procapsid Requires Both the Maturation Protease and the Portal Complex », Journal of Molecular Biology, vol. 428, nᵒ 1, p. 165-181, janv. 2016.
    Résumé : The 90-nm-diameter capsid of coliphage T5 is organized with T=13 icosahedral geometry and encloses a double-stranded DNA genome that measures 121kbp. Its assembly follows a path similar to that of phage HK97 but yielding a larger structure that includes 775 subunits of the major head protein, 12 subunits of the portal protein and 120 subunits of the decoration protein. As for phage HK97, T5 encodes the scaffold function as an N-terminal extension (∆-domain) to the major head protein that is cleaved by the maturation protease after assembly of the initial prohead I form and prior to DNA packaging and capsid expansion. Although the major head protein alone is sufficient to assemble capsid-like particles, the yield is poor and includes many deformed structures. Here we explore the role of both the portal and the protease in capsid assembly by generating constructs that include the major head protein and a combination of protease (wild type or an inactive mutant) and portal proteins and overexpressing them in Escherichia coli. Our results show that the inactive protease mutant acts to trigger assembly of the major head protein, probably through binding to the ∆-domain, while the portal protein regulates assembly into the correct T=13 geometry. A cryo-electron microscopy reconstruction of prohead I including inactivated protease reveals density projecting from the prohead interior surface toward its center that is compatible with the ∆-domain, as well as additional internal density that we assign as the inactivated protease. These results reveal complexity in T5 beyond that of the HK97 system.
    Mots-clés : capsid assembly, Cryoelectron Microscopy, DNA Mutational Analysis, Escherichia coli, maturation protease, portal, scaffolding, Siphoviridae, T5PHAG, tailed bacteriophage, Viral Proteins, VIRO, Virus Assembly.

  • G. Maarifi, Z. Hannoun, M. C. Geoffroy, F. El Asmi, K. Zarrouk, S. Nisole, D. Blondel, et M. K. Chelbi-Alix, « MxA Mediates SUMO-Induced Resistance to Vesicular Stomatitis Virus », Journal of Virology, vol. 90, nᵒ 14, p. 6598-6610, juill. 2016.
    Résumé : Multiple cellular pathways are regulated by small ubiquitin-like modifier (SUMO) modification, including ubiquitin-mediated proteolysis, signal transduction, innate immunity, and antiviral defense. In the study described in this report, we investigated the effects of SUMO on the replication of two members of the Rhabdoviridae family, vesicular stomatitis virus (VSV) and rabies virus (RABV). We show that stable expression of SUMO in human cells confers resistance to VSV infection in an interferon-independent manner. We demonstrate that SUMO expression did not alter VSV entry but blocked primary mRNA synthesis, leading to a reduction of viral protein synthesis and viral production, thus protecting cells from VSV-induced cell lysis. MxA is known to inhibit VSV primary transcription. Interestingly, we found that the MxA protein was highly stabilized in SUMO-expressing cells. Furthermore, extracts from cells stably expressing SUMO exhibited an increase in MxA oligomers, suggesting that SUMO plays a role in protecting MxA from degradation, thus providing a stable intracellular pool of MxA available to combat invading viruses. Importantly, MxA depletion in SUMO-expressing cells abrogated the anti-VSV effect of SUMO. Furthermore, SUMO expression resulted in interferon-regulatory factor 3 (IRF3) SUMOylation, subsequently decreasing RABV-induced IRF3 phosphorylation and interferon synthesis. As expected, this rendered SUMO-expressing cells more sensitive to RABV infection, even though MxA was stabilized in SUMO-expressing cells, since its expression did not confer resistance to RABV. Our findings demonstrate opposing effects of SUMO expression on two viruses of the same family, intrinsically inhibiting VSV infection through MxA stabilization while enhancing RABV infection by decreasing IFN induction. IMPORTANCE: We report that SUMO expression reduces interferon synthesis upon RABV or VSV infection. Therefore, SUMO renders cells more sensitive to RABV but unexpectedly renders cells resistant to VSV by blocking primary mRNA synthesis. Unlike the interferon-mediated innate immune response, intrinsic antiviral resistance is mediated by constitutively expressed restriction factors. Among the various anti-VSV restriction factors, only MxA is known to inhibit VSV primary transcription, and we show here that its expression does not alter RABV infection. Interestingly, MxA depletion abolished the inhibition of VSV by SUMO, demonstrating that MxA mediates SUMO-induced intrinsic VSV resistance. Furthermore, MxA oligomerization is known to be critical for its protein stability, and we show that higher levels of oligomers were formed in cells expressing SUMO than in wild-type cells, suggesting that SUMO may play a role in protecting MxA from degradation, providing a stable intracellular pool of MxA able to protect cells from viral infection.
    Mots-clés : RHABDO, VIRO.

  • M. A. Maroui, A. Callé, C. Cohen, N. Streichenberger, P. Texier, J. Takissian, A. Rousseau, N. Poccardi, J. Welsch, A. Corpet, L. Schaeffer, M. Labetoulle, et P. Lomonte, « Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment », PLoS pathogens, vol. 12, nᵒ 9, p. e1005834, sept. 2016.
    Résumé : Herpes simplex virus 1 (HSV-1) establishes latency in trigeminal ganglia (TG) sensory neurons of infected individuals. The commitment of infected neurons toward the viral lytic or latent transcriptional program is likely to depend on both viral and cellular factors, and to differ among individual neurons. In this study, we used a mouse model of HSV-1 infection to investigate the relationship between viral genomes and the nuclear environment in terms of the establishment of latency. During acute infection, viral genomes show two major patterns: replication compartments or multiple spots distributed in the nucleoplasm (namely "multiple-acute"). Viral genomes in the "multiple-acute" pattern are systematically associated with the promyelocytic leukemia (PML) protein in structures designated viral DNA-containing PML nuclear bodies (vDCP-NBs). To investigate the viral and cellular features that favor the acquisition of the latency-associated viral genome patterns, we infected mouse primary TG neurons from wild type (wt) mice or knock-out mice for type 1 interferon (IFN) receptor with wt or a mutant HSV-1, which is unable to replicate due to the synthesis of a non-functional ICP4, the major virus transactivator. We found that the inability of the virus to initiate the lytic program combined to its inability to synthesize a functional ICP0, are the two viral features leading to the formation of vDCP-NBs. The formation of the "multiple-latency" pattern is favored by the type 1 IFN signaling pathway in the context of neurons infected by a virus able to replicate through the expression of a functional ICP4 but unable to express functional VP16 and ICP0. Analyses of TGs harvested from HSV-1 latently infected humans showed that viral genomes and PML occupy similar nuclear areas in infected neurons, eventually forming vDCP-NB-like structures. Overall our study designates PML protein and PML-NBs to be major cellular components involved in the control of HSV-1 latency, probably during the entire life of an individual.
    Mots-clés : HERPES, VIRO.

  • L. Mouna, E. Hernandez, D. Bonte, R. Brost, L. Amazit, L. R. Delgui, W. Brune, A. P. Geballe, I. Beau, et A. Esclatine, « Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins », Autophagy, vol. 12, nᵒ 2, p. 327-342, 2016.
    Résumé : Autophagy is activated early after human cytomegalovirus (HCMV) infection but, later on, the virus blocks autophagy. Here we characterized 2 HCMV proteins, TRS1 and IRS1, which inhibit autophagy during infection. Expression of either TRS1 or IRS1 was able to block autophagy in different cell lines, independently of the EIF2S1 kinase, EIF2AK2/PKR. Instead, TRS1 and IRS1 interacted with the autophagy protein BECN1/Beclin 1. We mapped the BECN1-binding domain (BBD) of IRS1 and TRS1 and found it to be essential for autophagy inhibition. Mutant viruses that express only IRS1 or TRS1 partially controlled autophagy, whereas a double mutant virus expressing neither protein stimulated autophagy. A mutant virus that did not express IRS1 and expressed a truncated form of TRS1 in which the BBD was deleted, failed to control autophagy. However, this mutant virus had similar replication kinetics as wild-type virus, suggesting that autophagy inhibition is not critical for viral replication. In fact, using pharmacological modulators of autophagy and inhibition of autophagy by shRNA knockdown, we discovered that stimulating autophagy enhanced viral replication. Conversely, inhibiting autophagy decreased HCMV infection. Thus, our results demonstrate a new proviral role of autophagy for a DNA virus.
    Mots-clés : Autophagy, Beclin-1, BECN1, Cytomegalovirus, Cytomegalovirus Infections, eIF-2 Kinase, EIF2AK2/PKR, HeLa Cells, HERPES, Humans, IRS1, Male, Mutation, Protein Binding, Protein Domains, Recombination, Genetic, TRS1, Viral Proteins, VIRO.

  • J. Nikolic, A. Civas, Z. Lama, C. Lagaudrière-Gesbert, et D. Blondel, « Rabies Virus Infection Induces the Formation of Stress Granules Closely Connected to the Viral Factories », PLoS pathogens, vol. 12, nᵒ 10, p. e1005942, oct. 2016.
    Résumé : Stress granules (SGs) are membrane-less dynamic structures consisting of mRNA and protein aggregates that form rapidly in response to a wide range of environmental cellular stresses and viral infections. They act as storage sites for translationally silenced mRNAs under stress conditions. During viral infection, SG formation results in the modulation of innate antiviral immune responses, and several viruses have the ability to either promote or prevent SG assembly. Here, we show that rabies virus (RABV) induces SG formation in infected cells, as revealed by the detection of SG-marker proteins Ras GTPase-activating protein-binding protein 1 (G3BP1), T-cell intracellular antigen 1 (TIA-1) and poly(A)-binding protein (PABP) in the RNA granules formed during viral infection. As shown by live cell imaging, RABV-induced SGs are highly dynamic structures that increase in number, grow in size by fusion events, and undergo assembly/disassembly cycles. Some SGs localize in close proximity to cytoplasmic viral factories, known as Negri bodies (NBs). Three dimensional reconstructions reveal that both structures remain distinct even when they are in close contact. In addition, viral mRNAs synthesized in NBs accumulate in the SGs during viral infection, revealing material exchange between both compartments. Although RABV-induced SG formation is not affected in MEFs lacking TIA-1, TIA-1 depletion promotes viral translation which results in an increase of viral replication indicating that TIA-1 has an antiviral effect. Inhibition of PKR expression significantly prevents RABV-SG formation and favors viral replication by increasing viral translation. This is correlated with a drastic inhibition of IFN-B gene expression indicating that SGs likely mediate an antiviral response which is however not sufficient to fully counteract RABV infection.
    Mots-clés : RHABDO, VIRO.

  • G. Siracusano, A. Venuti, D. Lombardo, A. Mastino, A. Esclatine, et M. T. Sciortino, « Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells », Scientific Reports, vol. 6, p. 31302, août 2016.
    Résumé : Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.
    Mots-clés : HERPES, VIRO.


  • E. Baquero, A. A. V. Albertini, et Y. Gaudin, « Recent mechanistic and structural insights on class III viral fusion glycoproteins », Current Opinion in Structural Biology, vol. 33, p. 52-60, août 2015.
    Résumé : Enveloped viruses enter the cell by fusing their envelope with a cellular membrane. Fusion is catalyzed by conformational changes of viral glycoproteins from pre-fusion to post-fusion states. Structural studies have defined three classes of viral fusion glycoproteins. Class III comprises the fusion glycoproteins from rhabdoviruses (G), herpesviruses (gB), and baculoviruses (GP64). Although sharing the same fold, those glycoproteins exhibit striking differences in their modes of activation and interaction with the target membrane. Furthermore, for gB and GP64, only the post-fusion structure is known and the extent of their conformational change is still an unresolved issue. Further structural studies are therefore required to get a detailed insight in the working of those fusion machines.
    Mots-clés : Baculoviridae, Cell Membrane, Herpesviridae, Hydrogen-Ion Concentration, Protein Conformation, RHABDO, Rhabdoviridae, Viral Fusion Proteins, VIRO.

  • E. Baquero, A. A. Albertini, H. Raux, L. Buonocore, J. K. Rose, S. Bressanelli, et Y. Gaudin, « Structure of the Low pH Conformation of Chandipura Virus G Reveals Important Features in the Evolution of the Vesiculovirus Glycoprotein », PLOS Pathogens, vol. 11, nᵒ 3, p. e1004756, mars 2015.
    Mots-clés : B3S, Evolution, Molecular, Humans, Hydrogen-Ion Concentration, IMAPP, Nucleocapsid, Protein Structure, Tertiary, RHABDO, Vesiculovirus, Viral Fusion Proteins, VIRO.

  • F. Beilstein, L. Obiang, H. Raux, et Y. Gaudin, « Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2 », Journal of Virology, vol. 89, nᵒ 21, p. 11019-11029, nov. 2015.
    Résumé : The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. IMPORTANCE: The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy.
    Mots-clés : Base Sequence, Blotting, Western, Cysteine Endopeptidases, HeLa Cells, Humans, Immunoprecipitation, Molecular Sequence Data, Mutation, Protein Binding, RHABDO, Sequence Alignment, Sequence Analysis, DNA, Two-Hybrid System Techniques, Vesiculovirus, Viral Matrix Proteins, VIRO.

  • D. Blondel, G. Maarifi, S. Nisole, et M. K. Chelbi-Alix, « Resistance to Rhabdoviridae Infection and Subversion of Antiviral Responses », Viruses, vol. 7, nᵒ 7, p. 3675-3702, juill. 2015.
    Résumé : Interferon (IFN) treatment induces the expression of hundreds of IFN-stimulated genes (ISGs). However, only a selection of their products have been demonstrated to be responsible for the inhibition of rhabdovirus replication in cultured cells; and only a few have been shown to play a role in mediating the antiviral response in vivo using gene knockout mouse models. IFNs inhibit rhabdovirus replication at different stages via the induction of a variety of ISGs. This review will discuss how individual ISG products confer resistance to rhabdoviruses by blocking viral entry, degrading single stranded viral RNA, inhibiting viral translation or preventing release of virions from the cell. Furthermore, this review will highlight how these viruses counteract the host IFN system.
    Mots-clés : Animals, Humans, interferon, Interferons, ISG, rabies virus, RHABDO, Rhabdoviridae, Rhabdoviridae Infections, rhabdoviruses, vesicular stomatitis virus, VIRO.

  • Y. Chaban, R. Lurz, S. Brasilès, C. Cornilleau, M. Karreman, S. Zinn-Justin, P. Tavares, et E. V. Orlova, « Structural rearrangements in the phage head-to-tail interface during assembly and infection », Proceedings of the National Academy of Sciences, vol. 112, nᵒ 22, p. 7009-7014, juin 2015.
    Mots-clés : allosteric mechanism, B3S, Bacteriophage, Bacteriophages, Cryoelectron Microscopy, DNA gatekeeper, Genome, Viral, hybrid methods, INTGEN, Models, Molecular, PHAG+, Protein Conformation, viral infection, Viral Proteins, Viral Tail Proteins, VIRO, Virus Assembly, Virus Internalization.

  • V. Cvirkaite-Krupovic, R. Carballido-López, et P. Tavares, « Virus evolution toward limited dependence on nonessential functions of the host: the case of bacteriophage SPP1 », Journal of Virology, vol. 89, nᵒ 5, p. 2875-2883, mars 2015.
    Résumé : All viruses are obligate intracellular parasites and depend on certain host cell functions for multiplication. However, the extent of such dependence and the exact nature of the functions provided by the host cell remain poorly understood. Here, we investigated if nonessential Bacillus subtilis genes are necessary for multiplication of bacteriophage SPP1. Screening of a collection of 2,514 single-gene knockouts of nonessential B. subtilis genes yielded only a few genes necessary for efficient SPP1 propagation. Among these were genes belonging to the yuk operon, which codes for the Esat-6-like secretion system, including the SPP1 receptor protein YueB. In addition, we found that SPP1 multiplication was negatively affected by the absence of two other genes, putB and efp. The gene efp encodes elongation factor P, which enhances ribosome activity by alleviating translational stalling during the synthesis of polyproline-containing proteins. PutB is an enzyme involved in the proline degradation pathway that is required for infection in the post-exponential growth phase of B. subtilis, when the bacterium undergoes a complex genetic reprogramming. The putB knockout shortens significantly the window of opportunity for SPP1 infection during the host cell life cycle. This window is a critical parameter for competitive phage multiplication in the soil environment, where B. subtilis rarely meets conditions for exponential growth. Our results in combination with those reported for other virus-host systems suggest that bacterial viruses have evolved toward limited dependence on nonessential host functions. IMPORTANCE: A successful viral infection largely depends on the ability of the virus to hijack cellular machineries and to redirect the flow of building blocks and energy resources toward viral progeny production. However, the specific virus-host interactions underlying this fundamental transformation are poorly understood. Here, we report on the first systematic analysis of virus-host cross talk during bacteriophage infection in Gram-positive bacteria. We show that lytic bacteriophage SPP1 is remarkably independent of nonessential genes of its host, Bacillus subtilis, with only a few cellular genes being necessary for efficient phage propagation. We hypothesize that such limited dependence of the virus on its host results from a constant "evolutionary arms race" and might be much more widespread than currently thought.
    Mots-clés : Bacillus Phages, Bacillus subtilis, Gene Knockout Techniques, Genes, Bacterial, Genetic Testing, Host-Parasite Interactions, PHAG+, VIRO, Virus Internalization, Virus Replication.

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