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Title: Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase

Abstract

The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination.

Authors:
; ; ; ; ; ; ; ; ; ; ; ;  [1]
  1. Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)
Publication Date:
OSTI Identifier:
22360343
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 6; Other Information: PMCID: PMC2335076; PMID: 17554171; PUBLISHER-ID: en5233; OAI: oai:pubmedcentral.nih.gov:2335076; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; MATHEMATICAL SOLUTIONS; RESOLUTION; SOLUTIONS; TESTING

Citation Formats

Jabafi, Ilham, Selisko, Barbara, Coutard, Bruno, De Palma, Armando M., Neyts, Johan, Egloff, Marie-Pierre, Grisel, Sacha, Dalle, Karen, Campanacci, Valerie, Spinelli, Silvia, Cambillau, Christian, Canard, Bruno, and Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107020416.
Jabafi, Ilham, Selisko, Barbara, Coutard, Bruno, De Palma, Armando M., Neyts, Johan, Egloff, Marie-Pierre, Grisel, Sacha, Dalle, Karen, Campanacci, Valerie, Spinelli, Silvia, Cambillau, Christian, Canard, Bruno, & Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr. Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase. United Kingdom. doi:10.1107/S1744309107020416.
Jabafi, Ilham, Selisko, Barbara, Coutard, Bruno, De Palma, Armando M., Neyts, Johan, Egloff, Marie-Pierre, Grisel, Sacha, Dalle, Karen, Campanacci, Valerie, Spinelli, Silvia, Cambillau, Christian, Canard, Bruno, and Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr. Fri . "Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase". United Kingdom. doi:10.1107/S1744309107020416.
@article{osti_22360343,
title = {Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase},
author = {Jabafi, Ilham and Selisko, Barbara and Coutard, Bruno and De Palma, Armando M. and Neyts, Johan and Egloff, Marie-Pierre and Grisel, Sacha and Dalle, Karen and Campanacci, Valerie and Spinelli, Silvia and Cambillau, Christian and Canard, Bruno and Gruez, Arnaud, E-mail: arnaud.gruez@maem.uhp-nancy.fr},
abstractNote = {The first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 Å resolution and that were suitable for structure determination.},
doi = {10.1107/S1744309107020416},
journal = {Acta Crystallographica. Section F},
number = Pt 6,
volume = 63,
place = {United Kingdom},
year = {Fri Jun 01 00:00:00 EDT 2007},
month = {Fri Jun 01 00:00:00 EDT 2007}
}
  • Cited by 2
  • In adolescent CD-1 male mice inoculated with a myocarditic coxsackievirus B3 (CVB3m) acute focal lesions containing necrotic myocytes, infiltrating mononuclear cells, and fibroblasts develop. With the use of an in situ immune autoradiographic method with rat monoclonal antibodies (MAb) and an /sup 35/S-labeled antibody, viral antigens were detected outside of lesions. Macrophages, T lymphocytes, and natural killer (NK) cells were identified within myocarditic lesions during the acute phase of the disease. Macrophages detected by anti-Mac-1 MAb were in focal areas within myocarditic lesions on Days 4-7 after inoculation. T lymphocytes were detected in myocarditic lesions on Days 4-10, with MAbmore » to Thy-1 and Lyt-1 antigens showing diffuse reaction patterns, suggesting random distribution of these cells in lesions. Focal areas of reactivity were detected with MAbs to L3T4 and Lyt-2 antigens, suggesting clusters of helper and cytotoxic/suppressor T lymphocytes, respectively. NK cells were presumptively detected by asialo GM1 surface marker in lesions at all times. The presence of activated NK cells in lesions was confirmed by assay of mechanically dissociated heart tissues on Day 8. These data describe the temporal sequence and identity of leukocytes entering into CVB3-induced focal myocarditic lesions during the acute phase of disease in CD-1 mice.« less
  • Coxsackievirus B3 (CVB3) is nonenveloped and has a single-stranded positive-sense RNA genome. CVB3 induces myocarditis and ultimately dilated cardiomyopathy. Although there are mounting evidences of an interaction between CVB3 particles and the cellular receptors, coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF), very little is known about the mechanisms of internalization and trafficking. In the present study, we used the CVB3 H3 strain, which is CAR-dependent but DAF-independent Woodruff variant and found that during entry, CVB3 particles were colocalized in clathrin, after interacting primarily with CAR, which was not recycled to the plasma membrane. We also found that CVB3more » internalization was dependent on the function of dynamin, a large GTPase that has an essential role in endocytosis. Heat-shock cognate protein, Hsc70, which acts as a chaperone in the release of coat proteins from clathrin-coated vesicles (CCV), played a role in CVB3 trafficking processes. Moreover, endosomal acidification was crucial for CVB3 endocytosis. Finally, CVB3 was colocalized in early endosome autoantigen 1 (EEA1) molecules, which are involved in endosome-endosome tethering and fusion. In conclusion, these data together indicate that CVB3 uses clathrin-mediated endocytosis and is transcytosed to early endosomes.« less
  • The capsid proteins of some RNA viruses can translocate to the nucleus and interfere with cellular phenotypes. In this study we found that the VP1 capsid protein of coxsackievirus B3 (CVB3) was dominantly localized in the nucleus of the cells transfected with VP1-expressing plasmid. The VP1 nuclear localization also occurred in the cells infected with CVB3. Truncation analysis indicated that the VP1 nuclear localization sequence located near the C-terminal. The substitution of His220 with threonine completely abolished its translocation. The VP1 proteins of other CVB types might have the nuclear localization potential because this region was highly conserved. Moreover, themore » VP1 nuclear localization induced cell cycle deregulation, including a prolonged S phase and shortened G2-M phase. Besides these findings, we also found a domain between Ala72 and Phe106 that caused the VP1 truncates dotted distributed in the cytoplasm. Our results suggest a new pathogenic mechanism of CVB. - Highlights: Black-Right-Pointing-Pointer The VP1 protein of coxsackievirus B3 can specifically localize in the nucleus. Black-Right-Pointing-Pointer The nuclear localization signal of coxsackievirus B3 VP1 protein locates near its C-terminal. Black-Right-Pointing-Pointer The VP1 nuclear localization of coxsackievirus B3 can deregulate cell cycle. Black-Right-Pointing-Pointer There is a domain in the VP1 that determines it dotted distributed in the cytoplasm.« less