The interaction between the measles virus nucleoprotein and the Interferon Regulator Factor 3 relies on a specific cellular environment

Virology Journal BioMed Central Research Open Access The interaction between the measles virus nucleoprotein and the Interferon Regulator Factor 3 relies on a specific cellular environment Matteo Colombo1,2, Jean-Marie Bourhis1,3, Celia Chamontin4, Carine Soriano4, Stéphanie Villet4, Stéphanie Costanzo1, Marie Couturier1, Valérie Belle5, André Fournel5, Hervé Darbon1, Denis Gerlier*4 and Sonia Longhi*1 Address: 1Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS et Universités Aix-Marseille I et II, 163 Avenue de Luminy, Case 932, 13288 Marseille Cedex 09, France, 2Dept of Biomolecular Sciences and Biotechnology, Universita' degli Studi di Milano, Via Celoria, 26. I-20133 Milan, Italy, 3Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS Université de Lyon, 7, passage du Vercors, 69 367 Lyon cedex 7, France, 4VirPatH, FRE 3011, CNRS and Université Lyon 1, Faculté de Médecine RTH Laennec, 69372, Lyon, France and 5Bioénergétique et Ingénierie des Protéines, UPR 9036 CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex, France, and Université Aix-Marseille I, 3 place Victor Hugo 13331, Marseille, Cedex 3, France Email: Matteo Colombo - ; Jean-Marie Bourhis - ; Celia Chamontin - ; Carine Soriano - ; Stéphanie Villet - ; Stéphanie Costanzo - ; Marie Couturier - ; Valérie Belle - ; André Fournel - ; Hervé Darbon - ; Denis Gerlier* - ; Sonia Longhi* - * Corresponding authors Published: 15 May 2009 Virology Journal 2009, 6:59 doi:10.1186/1743-422X-6-59 Received: 11 March 2009 Accepted: 15 May 2009 This article is available from: http://www.virologyj.com/content/6/1/59 © 2009 Colombo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The genome of measles virus consists of a non-segmented single-stranded RNA molecule of negative polarity, which is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. The N protein possesses an intrinsically disordered C-terminal domain (aa 401–525, NTAIL) that is exposed at the surface of the viral nucleopcapsid. Thanks to its flexible nature, NTAIL interacts with several viral and cellular partners. Among these latter, the Interferon Regulator Factor 3 (IRF-3) has been reported to interact with N, with the interaction having been mapped to the regulatory domain of IRF-3 and to NTAIL. This interaction was described to lead to the phosphorylation-dependent activation of IRF-3, and to the ensuing activation of the pro-immune cytokine RANTES gene. Results: After confirming the reciprocal ability of IRF-3 and N to be co-immunoprecipitated in 293T cells, we thoroughly investigated the NTAIL-IRF-3 interaction using a recombinant, monomeric form of the regulatory domain of IRF-3. Using a large panel of spectroscopic approaches, including circular dichroism, fluorescence spectroscopy, nuclear magnetic resonance and electron paramagnetic resonance spectroscopy, we failed to detect any direct interaction between IRF-3 and either full-length N or NTAIL under conditions where these latter interact with the C-terminal X domain of the viral phosphoprotein. Furthermore, such interaction was neither detected in E. coli nor in a yeast two hybrid assay. Conclusion: Altogether, these data support the requirement for a specific cellular environment, such as that provided by 293T human cells, for the NTAIL-IRF-3 interaction to occur. This dependence from a specific cellular context likely reflects the requirement for a human or mammalian cellular co-factor. Page 1 of 17 (page number not for citation purposes) Virology Journal 2009, 6:59 Background Measles virus (MeV) is an enveloped RNA virus within the Morbillivirus genus of the Paramyxoviridae family. Its nonsegmented, negative-sense, single-stranded RNA genome is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. Transcription and replication are carried out onto this N:RNA complex by the viral polymerase complex which consists of two components, the large protein (L) and the phosphoprotein (P) (reviewed in [1]). MeV N consists of two regions: a structured N-terminal moiety, NCORE (aa 1–400), which contains all the regions necessary for self-assembly and RNA-binding [2,3], and a C-terminal domain, NTAIL(aa 401–525) that is intrinsically unstructured [4] and is exposed at the surface of the viral nucleocapsid [2,5,6]. Intrinsically disordered proteins (IDPs) or protein domains lack highly populated and uniform secondary and tertiary structure under physiological conditions but fulfill essential biological functions [7-19]. Since NTAIL is intrinsically flexible and is exposed at the surface of the viral nucleocapsid, it interacts with various partners, including the viral P protein [3,4] and host cell proteins such as the major inducible heat shock protein (Hsp72) [20,21], and the yet uncharacterized Nucleoprotein Receptor (NR) [22,23]. In addition, it has also been reported to interact with the Interferon Regulator Factor 3 (IRF-3) [24]. IRF-3 is ubiquitously expressed as a stable latent transactivator of the cellular innate immune response [25]. It belongs to the family of interferon regulatory factors (IRF) and acts as a transactivator for the interferon-β (IFN-β) and various pro-inflammatory cytokine genes. All mammalian IRFs share a conserved N-terminal DNA binding domain (DBD) and a C-terminal interferon association domain (IAD). IRF-3 consists of a DBD (aa 1–110), of a proline-rich region (PRR, aa 112–174), followed by the IAD (aa 175–384) and by a serine-rich region (SRR, aa 385–427) (Figure 1A). The seminal and unique observation that MeV N activates IRF-3 to induce CCL5 (also called RANTES), a pro-inflammatory cytokine, but not IFN-β, was done by the Hiscott's group ?[24]. After MeV infection, IRF-3 was phosphorylated at the key Ser385 and Ser386 residues, and this form was able to bind to the interferon sensitive response element of ISG15 in complex with CREB binding protein in vitro. Activation of IRF-3, which required active MeV transcription, was also mimicked by the transient expression of the N protein [24]. Moreover, IRF-3 and a cellular kinase could be co-immunoprecipitated with N [24]. From these data it was assumed that MeV N physically http://www.virologyj.com/content/6/1/59 interacts with IRF-3 and induces the phosphorylation of the latter by recruiting the kinase. Phosphorylation of IRF3 would then lead to IRF-3 homo-dimerisation, followed by IRF-3 nuclear import and transactivation of a selective set of pro-inflammatory cytokines [24]. Using deletion constructs and co-immunoprecipitation studies, the IRF-3 binding region was grossly mapped to NTAIL (residues 415–523), while the N binding region within IRF-3 was mapped to residu (...truncated)