Abstract
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.
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Acknowledgements
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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Financial grants and infrastructure 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) and by a grant to Dr Schoehn Guy, from the electron microscopy platform of the Integrated Structural Biology of Grenoble (ISBG, UMS 3518). Equipments used to perform measures of biophysical parameters were from National Synchrotron Light Laboratory (LNLS, Brazil) and Central Experimental Multiusuário da Universidade Federal do ABC (CEM/UFABC). This work was developed with Grants obtained from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), by Márcia Aparecida Sperança number # 2009/11347-6 and by Wanius José Garcia number # 2015/02897-3. Fellowships Aline Diniz Cabral and Viviam Moura da Silva received fellowship from FAPESP, numbers # 2013/26096-4 and 2012/03503-0, respectively. Juliana Londoño Murillo, Mabel Uehara and Juliete Vitorino dos Santos received fellowship from UFABC, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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726_2018_2560_MOESM1_ESM.tif
Characterization of OROV-rNP by dynamic light scattering (DLS) at pH 7.5 and 20 °C. (A) Correlation time. (B) The hydrodynamic radius (R s ) determined for the OROV-rNP was of RS = 6.0 ± 0.5 nm. 1 (TIFF 191 kb)
726_2018_2560_MOESM2_ESM.tif
Transmission Electron Microscopy. (Top) Negatively stained transmission electron micrograph. Scale bar 100 nm. (Bottom) Semi-automatically selected rNP particles. Scale bar 30 nm. 2 (TIFF 1773 kb)
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Murillo, J.L., Cabral, A.D., Uehara, M. et al. Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro. Amino Acids 50, 711–721 (2018). https://doi.org/10.1007/s00726-018-2560-4
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DOI: https://doi.org/10.1007/s00726-018-2560-4