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Publications Département Virologie


  • 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, glycoprotein, IMAPP, intermediate structures, membrane fusion, 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, 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.

  • 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, 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.

  • 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, 2017.
    Mots-clés : B3S, FAAM, INTGEN, T5PHAG, VIRO.

  • 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.
    Mots-clés : B3S, FAAM, INTGEN, T5PHAG, VIRO.


  • 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, 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, 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, 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, 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.

  • D. Liger, L. Mora, N. Lazar, S. Figaro, J. Henri, N. Scrima, R. H. Buckingham, H. van Tilbeurgh, V. Heurgué-Hamard, et M. Graille, « Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein », Nucleic Acids Research, vol. 44, nᵒ 3, p. 1482-1482, févr. 2016.
    Mots-clés : B3S, FAAM, RHABDO, VIRO.

  • 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, 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, 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, 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, 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, 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, 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, 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, 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.

  • B. Fouquet, J. Nikolic, F. Larrous, H. Bourhy, C. Wirblich, C. Lagaudrière-Gesbert, et D. Blondel, « Focal adhesion kinase is involved in rabies virus infection through its interaction with viral phosphoprotein P », Journal of Virology, vol. 89, nᵒ 3, p. 1640-1651, 2015.
    Résumé : The rabies virus (RABV) phosphoprotein P is a multifunctional protein: it plays an essential role in viral transcription and replication, and in addition, RABV P has been identified as an interferon antagonist. Here, a yeast two-hybrid screen revealed that RABV P interacts with the focal adhesion kinase (FAK). The binding involved the 106-to-131 domain, corresponding to the dimerization domain of P and the C-terminal domain of FAK containing the proline-rich domains PRR2 and PRR3. The P-FAK interaction was confirmed in infected cells by coimmunoprecipitation and colocalization of FAK with P in Negri bodies. By alanine scanning, we identified a single mutation in the P protein that abolishes this interaction. The mutant virus containing a substitution of Ala for Arg in position 109 in P (P.R109A), which did not interact with FAK, is affected at a posttranscriptional step involving protein synthesis and viral RNA replication. Furthermore, FAK depletion inhibited viral protein expression in infected cells. This provides the first evidence of an interaction of RABV with FAK that positively regulates infection. IMPORTANCE: Rabies virus exhibits a small genome that encodes a limited number of viral proteins. To maintain efficient virus replication, some of them are multifunctional, such as the phosphoprotein P. We and others have shown that P establishes complex networks of interactions with host cell components. These interactions have revealed much about the role of P and about host-pathogen interactions in infected cells. Here, we identified another cellular partner of P, the focal adhesion kinase (FAK). Our data shed light on the implication of FAK in RABV infection and provide evidence that P-FAK interaction has a proviral function.
    Mots-clés : Animals, Cell Line, DNA Mutational Analysis, Focal Adhesion Protein-Tyrosine Kinases, Host-Pathogen Interactions, Humans, Immunoprecipitation, Inclusion Bodies, Viral, Microscopy, Confocal, Mutagenesis, Site-Directed, Phosphoproteins, Protein Binding, Protein Interaction Mapping, rabies virus, RHABDO, Two-Hybrid System Techniques, Viral Structural Proteins, VIRO, Virus Replication.

  • C. Garcia-Doval, J. R. Castón, D. Luque, M. Granell, J. M. Otero, A. L. Llamas-Saiz, M. Renouard, P. Boulanger, et M. J. van Raaij, « Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone », Viruses, vol. 7, nᵒ 12, p. 6424-6440, 2015.
    Résumé : Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970-1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263-1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.
    Mots-clés : bacterial viruses, Caudovirales, crystallography, Crystallography, X-Ray, infection, J0101, Models, Molecular, Molecular Chaperones, Mutant Proteins, Protein Conformation, Siphoviridae, T5PHAG, Viral Tail Proteins, VIRO, Virus Attachment.

  • M. Gratia, E. Sarot, P. Vende, A. Charpilienne, C. H. Baron, M. Duarte, S. Pyronnet, et D. Poncet, « Rotavirus NSP3 Is a Translational Surrogate of the Poly(A) Binding Protein-Poly(A) Complex », Journal of Virology, vol. 89, nᵒ 17, p. 8773-8782, 2015.
    Résumé : Through its interaction with the 5' translation initiation factor eIF4G, poly(A) binding protein (PABP) facilitates the translation of 5'-capped and 3'-poly(A)-tailed mRNAs. Rotavirus mRNAs are capped but not polyadenylated, instead terminating in a 3' GACC motif that is recognized by the viral protein NSP3, which competes with PABP for eIF4G binding. Upon rotavirus infection, viral, GACC-tailed mRNAs are efficiently translated, while host poly(A)-tailed mRNA translation is, in contrast, severely impaired. To explore the roles of NSP3 in these two opposing events, the translational capabilities of three capped mRNAs, distinguished by either a GACC, a poly(A), or a non-GACC and nonpoly(A) 3' end, have been monitored after electroporation of cells expressing all rotavirus proteins (infected cells) or only NSP3 (stably or transiently transfected cells). In infected cells, we found that the magnitudes of translation induction (GACC-tailed mRNA) and translation reduction [poly(A)-tailed mRNA] both depended on the rotavirus strain used but that translation reduction not genetically linked to NSP3. In transfected cells, even a small amount of NSP3 was sufficient to dramatically enhance GACC-tailed mRNA translation and, surprisingly, to slightly favor the translation of both poly(A)- and nonpoly(A)-tailed mRNAs, likely by stabilizing the eIF4E-eIF4G interaction. These data suggest that NSP3 is a translational surrogate of the PABP-poly(A) complex; therefore, it cannot by itself be responsible for inhibiting the translation of host poly(A)-tailed mRNAs upon rotavirus infection. IMPORTANCE: To control host cell physiology and to circumvent innate immunity, many viruses have evolved powerful mechanisms aimed at inhibiting host mRNA translation while stimulating translation of their own mRNAs. How rotavirus tackles this challenge is still a matter of debate. Using rotavirus-infected cells, we show that the magnitude of cellular poly(A) mRNA translation differs with respect to rotavirus strains but is not genetically linked to NSP3. Using cells expressing rotavirus NSP3, we show that NSP3 alone not only dramatically enhances rotavirus-like mRNA translation but also enhances poly(A) mRNA translation rather than inhibiting it, likely by stabilizing the eIF4E-eIF4G complex. Thus, the inhibition of cellular polyadenylated mRNA translation during rotavirus infection cannot be attributed solely to NSP3 and is more likely the result of global competition between viral and host mRNAs for the cellular translation machinery.
    Mots-clés : Animals, Cell Line, Cricetinae, Electroporation, Eukaryotic Initiation Factor-4E, Eukaryotic Initiation Factor-4G, HeLa Cells, Humans, Macaca mulatta, Poly A, Poly(A)-Binding Proteins, Polyadenylation, Protein Binding, Protein Biosynthesis, RNA, Messenger, RNA, Viral, ROTA, Rotavirus, Rotavirus Infections, Transfection, Viral Nonstructural Proteins, VIRO.

  • C. Langlois, S. Ramboarina, A. Cukkemane, I. Auzat, B. Chagot, B. Gilquin, A. Ignatiou, I. Petitpas, E. Kasotakis, M. Paternostre, H. E. White, E. V. Orlova, M. Baldus, P. Tavares, et S. Zinn-Justin, « Bacteriophage SPP1 Tail Tube Protein Self-assembles into β-Structure-rich Tubes », Journal of Biological Chemistry, vol. 290, nᵒ 6, p. 3836-3849, févr. 2015.
    Mots-clés : Amino Acid Sequence, B3S, Bacteriophage, Bacteriophages, Electron Microscopy, Fourier Transform IR (FTIR), IMAPP, INTGEN, Molecular Sequence Data, PHAG+, Protein Folding, Protein Structure, Tertiary, Solid State NMR, Tail Tube, Tertiary Structure, Viral Proteins, Virion, VIRO, Virus Assembly.

  • N. Scrima, J. Lepault, Y. Boulard, D. Pasdeloup, S. Bressanelli, et S. Roche, « Insights into herpesvirus tegument organization from structural analyses of the 970 central residues of HSV-1 UL36 protein », The Journal of Biological Chemistry, vol. 290, nᵒ 14, p. 8820-8833, 2015.
    Résumé : The tegument of all herpesviruses contains a capsid-bound large protein that is essential for multiple viral processes, including capsid transport, decapsidation at the nuclear pore complex, particle assembly, and secondary envelopment, through mechanisms that are still incompletely understood. We report here a structural characterization of the central 970 residues of this protein for herpes simplex virus type 1 (HSV-1 UL36, 3164 residues). This large fragment is essentially a 34-nm-long monomeric fiber. The crystal structure of its C terminus shows an elongated domain-swapped dimer. Modeling and molecular dynamics simulations give a likely molecular organization for the monomeric form and extend our findings to alphaherpesvirinae. Hence, we propose that an essential feature of UL36 is the existence in its central region of a stalk capable of connecting capsid and membrane across the tegument and that the ability to switch between monomeric and dimeric forms may help UL36 fulfill its multiple functions.
    Mots-clés : Amino Acid Sequence, B3S, Cell Line, Transformed, Crystal Structure, Dimerization, Herpesvirus, Herpesvirus 1, Human, HSV1, Humans, IMAPP, Molecular Sequence Data, Protein Conformation, Sequence Homology, Amino Acid, Structural Biology, Tegument, ul36, Viral Proteins, VIRO, VIROEM, Virus Assembly, Virus Structure, vp1/2.

  • F. Thiam, A. Charpilienne, D. Poncet, E. Kohli, et C. Basset, « B subunits of cholera toxin and thermolabile enterotoxin of Escherichia coli have similar adjuvant effect as whole molecules on rotavirus 2/6-VLP specific antibody responses and induce a Th17-like response after intrarectal immunization », Microbial Pathogenesis, vol. 89, p. 27-34, 2015.
    Résumé : The purpose of this study was to evaluate the adjuvant effect of the B subunits of cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) by the intrarectal route of immunization and compare them to the whole molecules CT and LT-R192G, a non toxic mutant of LT, using 2/6-VLP as an antigen, in mice. All molecules induced similar antigen specific antibody titers in serum and feces, whereas different T cell profiles were observed. CTB and LTB, conversely to CT and LT-R192G, did not induce detectable production of IL-2 by antigen specific T cells. Moreover, CTB, conversely to LT-R192G, CT and LTB, did not induce antigen specific CD4+CD25+Foxp3- and Foxp3+ T cells, thus showing different effects between the B subunits themselves. However, all molecules induced an antigen specific Th17 response. In conclusion, B subunits are potent adjuvants on B cell responses by the intrarectal route. Although their impact on T cell responses are different, all molecules induce a 2/6-VLP-specific Th17 T cell response that may play a major role in helping B cell responses and thus in adjuvanticity and protection.
    Mots-clés : Adjuvants, Immunologic, Administration, Rectal, Animals, Antibodies, Viral, Antibody Formation, B subunit, Bacterial Toxins, Cholera Toxin, Enterotoxins, Escherichia coli Proteins, Feces, Immunization, Immunologic Memory, Interleukin-2, Intrarectal, LT-R192G, Mice, ROTA, Rotavirus, Rotavirus Vaccines, Th17 Cells, Vaccines, Virus-Like Particle, VIRO.
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