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Accueil > Départements > Virologie > Pascale BOULANGER : Bactériophage T5

Publications

2017

2016


  • 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.

  • 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.

2015


  • 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.
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Publications avant 2015

Zivanovic Y, Confalonieri F, Ponchon L, Lurz R, Chami M, Flayhan A, Renouard M, Huet A, Decottignies P, Davidson AR, Breyton C, Boulanger P. (2014) Insights into bacteriophage T5 structure from the analysis of its morphogenesis genes and protein components. J Virol 88:1162-1174

Flayhan A, Vellieux FM, Lurz R, Maury O, Contreras-Martel C, Girard E, Boulanger P, Breyton C. (2014) Crystal structure of pb9, the distal tail protein of bacteriophage T5 : A conserved structural motif among all siphophages. J Virol 88:820-828
Garcia-Doval C, Luque D, Castón JR, Boulanger P, van Raaij MJ. (2013)

Crystallization of the C-terminal domain of the bacteriophage T5 L-shaped fibre. Acta Cryst. F69, 1363–1367

C. Breyton, A. Flayhan, F. Gabel, M. Lethier, G. Durand, P. Boulanger, M. Chami, C. Ebel (2013) Assessing the conformational changes of pb5, the receptor-binding protein of Phage T5, upon binding to its Escherichia coli receptor FhuA. J Biol Chem 288:30763-72

Preux O, Durand D, Huet A, Conway JF, Bertin A, Boulogne C, Drouin-Wahbi J, Trévarin D, Pérez J, Vachette P, Boulanger P. (2013) A two-state cooperative expansion converts the procapsid shell of bacteriophage T5 into a highly stable capsid isomorphous to the final virion head. J. Mol. Biol. 425 :1999-2014

Flayhan A, Wien F, Paternostre M, Boulanger P, Breyton C. (2012) New insights into pb5, the receptor binding protein of bacteriophage T5, and its interaction with its E. coli receptor FhuA. Biochimie 94:1982-89

Chiaruttini N, de Frutos M, Augarde E, Boulanger P, Letellier L, Viasnoff V. (2010) Is the in vitro ejection of bacteriophage DNA quasistatic ? A bulk to single virus study. Biophys. J. 99, 447-55

Huet A, Conway JF, Letellier L, Boulanger P. (2010) In vitro assembly of the T=13 procapsid of bacteriophage T5 with its scaffolding domain. J Virol 84, 9350-8

Braun T., Ghatkesar M. K., Backmann N., Grange W., Boulanger P., Letellier L., Lang H−P., Bietsch A., Gerber C. and Hegner, M. (2009). Quantitative, time-resolved measurement of membrane protein−ligand interactions using microcantilever array sensors. Nature Nanotechnology 4, 179−85

Boulanger, P. Jacquot P., Plançon, L., Chami, M., Engel, A., Parquet, C., Herbeuval, C., Letellier, L. (2008) Phage T5 straight fiber is a multifunctional protein acting as a tape measure and carrying fusogenic and muralytic activities. J. Biol. Chem, 16, 13556-13564

G. Effantin, P. Boulanger, E. Neumann, L. Letellier and J. F. Conway (2006) Bacteriophage T5 structure reveals similarities with HK97 & T4 suggesting evolutionary relationships. J. Mol. Biol. 361, 1003-1034

Ponchon, L., Boulanger, P., Labesse, G. & Letellier, L. (2006) Bacteriophage Terminases Belongs to the Resolvase/Integrase/ribonuclease H Superfamily J.Biol.Chem. 281, 5829-5836

L. Ponchon, S. Mangenot, P. Boulanger and L. Letellier (2005). Encapsidation and transfer of phage DNA into host cells : from in vivo to single particles studies. Biochim Biophys Acta. 1724, 225-261

Letellier L., Boulanger P., Plançon L., Jacquot P., and Santamaria M. (2004) Main features on tailed phage, host recognition and DNA uptake. Front. Biosci. 9, 1228-1239.

Letellier L., Boulanger P., de Frutos M., and Jacquot P. (2003). Channeling of phage DNA through membranes : from in vivo to in vitro. Res. Microbiol. 154, 283-287

L. Plançon , C. Janmot, M. le Maire , M. Desmadril, M. Bonhivers, L. Letellier and P. Boulanger (2002) Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5. J. Mol. Biol. 318, 557-569

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