Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro
Oropouche virus (OROV) is the unique known human pathogen belonging to serogroup Simbu of Orthobunyavirus genus and Bunyaviridae family. OROV is transmitted by wild mosquitoes species to sloths, rodents, monkeys and birds in sylvatic environment, and by midges (Culicoides paraensis and Culex quinquefasciatus) to man causing explosive outbreaks in urban locations. OROV infection causes dengue fever-like symptoms and in few cases, can cause clinical symptoms of aseptic meningitis. OROV contains a tripartite negative RNA genome encapsidated by the viral nucleocapsid protein (NP), which is essential for viral genome encapsidation, transcription and replication. Here, we reported the first study on the structural properties of a recombinant NP from human pathogen Oropouche virus (OROV–rNP). OROV–rNP was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Purified OROV–rNP was analyzed using a series of biophysical tools and molecular modeling. The results showed that OROV–rNP formed stable oligomers in solution coupled with endogenous E. coli nucleic acids (RNA) of different sizes. Finally, electron microscopy revealed a total of eleven OROV–rNP oligomer classes with tetramers (42%) and pentamers (43%) the two main populations and minor amounts of other bigger oligomeric states, such as hexamers, heptamers or octamers. The different RNA sizes and nucleotide composition may explain the diversity of oligomer classes observed. Besides, structural differences among bunyaviruses NP can be used to help in the development of tools for specific diagnosis and epidemiological studies of this group of viruses.
KeywordsOropouche Viral protein Nucleoprotein–RNA complex Arboviruses
This work used the platforms of the the Grenoble Instruct-ERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). The electron microscope facility is supported by the Rhône-Alpes Region, the Fondation Recherche Medicale (FRM), the fonds FEDER, the Centre National de la Recherche Scientifique (CNRS), the CEA, the University of Grenoble, EMBL, and the GIS-Infrastrutures en Biologie Sante et Agronomie (IBISA). We thank Dr Schoehn Guy, from the electron microscopy platform of the Integrated Structural Biology of Grenoble (ISBG, UMS 3518). We would like to thank the National Synchrotron Light Laboratory (LNLS, Brazil) and Central Experimental Multiusuário da Universidade Federal do ABC (CEM/UFABC); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support via grants # 2009/11347-6 (MAS), # 2015/02897-3 (WG), and fellowships # 2013/26096-4 (ADC), # 2012/03503-0 (VMS); UFABC, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowship to JLM, MU and JVS.
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All authors of this work declare that have no potential conflict of interest and that there is no financial, consultant, institutional or other relationships that might lead to bias or conflicts of interest in this research. Financial grants, infrastructure and fellowships supporting this work are described below.
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Consent to submit this work has been received explicitly from all co-authors, as well as from the institute and the university where the work has been carried out. All authors contributed to the scientific work and, therefore, share collective responsibility and accountability for the results.
- Boshra H, Lorenzo G, Rodriguez F, Brun A (2011) A DNA vaccine encoding ubiquitinated Rift Valley fever virus nucleoprotein provides consistent immunity and protects IFNAR(−/−) mice upon lethal virus challenge. Vaccine 29:4469–4475. https://doi.org/10.1016/j.vaccine.2011.04.043 CrossRefPubMedGoogle Scholar
- Hashem GM, Pham L, Vaughan MR, Gray DM (1998) Hybrid oligomer duplexes formed with phosphorothioate DNAs: CD spectra and melting temperatures of S-DNA.RNA hybrids are sequence-dependent but consistent with similar heteronomous conformations. Biochemistry 37:61–72. https://doi.org/10.1021/bi9713557 CrossRefPubMedGoogle Scholar
- Soldan SS, Gonzalez-Scarano F (2014) The bunyaviridae handbook of clinical neurology 123:449–463. https://doi.org/10.1016/B978-0-444-53488-0.00021-3 CrossRefPubMedGoogle Scholar
- Travassos da Rosa JF, de Souza WM, Pinheiro FP, Figueiredo ML, Cardoso JF, Acrani GO, Nunes MRT (2017) Oropouche virus: clinical, epidemiological, and molecular aspects of a neglected orthobunyavirus. Am J Trop Med Hyg 96:1019–1030. https://doi.org/10.4269/ajtmh.16-0672 PubMedPubMedCentralGoogle Scholar
- Whitehouse CA, Kuhn JH, Wada J, Ergunay K (2015) Family bunyaviridae. In: Shapshak P, Sinnott JT, Somboonwit C, Kuhn JH (eds) Global virology I—identifying and investigating viral diseases, vol 1. Springer, New York, p 847. https://doi.org/10.1007/978-1-4939-2410-3_10
- Yanase T, Kato T, Aizawa M, Shuto Y, Shirafuji H, Yamakawa M, Tsuda T (2012) Genetic reassortment between Sathuperi and Shamonda viruses of the genus Orthobunyavirus in nature: implications for their genetic relationship to Schmallenberg virus. Adv Virol 157:1611–1616. https://doi.org/10.1007/s00705-012-1341-8 Google Scholar