Amino Acids

pp 1–11 | Cite as

Nucleoprotein from the unique human infecting Orthobunyavirus of Simbu serogroup (Oropouche virus) forms higher order oligomers in complex with nucleic acids in vitro

  • Juliana Londoño Murillo
  • Aline Diniz Cabral
  • Mabel Uehara
  • Viviam Moura da Silva
  • Juliete Vitorino dos Santos
  • João Renato Carvalho Muniz
  • Leandro Farias Estrozi
  • Daphna Fenel
  • Wanius Garcia
  • Márcia Aparecida Sperança
Original Article


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.


Oropouche 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.

Compliance with ethical standards

Conflict of interest

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.

Human and animal rights statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

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.

Supplementary material

726_2018_2560_MOESM1_ESM.tif (191 kb)
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 (1.7 mb)
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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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  • Juliana Londoño Murillo
    • 1
  • Aline Diniz Cabral
    • 1
  • Mabel Uehara
    • 1
  • Viviam Moura da Silva
    • 2
  • Juliete Vitorino dos Santos
    • 1
  • João Renato Carvalho Muniz
    • 3
  • Leandro Farias Estrozi
    • 4
  • Daphna Fenel
    • 4
  • Wanius Garcia
    • 2
  • Márcia Aparecida Sperança
    • 1
  1. 1.Centro de Ciências Naturais e HumanasUniversidade Federal do ABCSão PauloBrazil
  2. 2.Centro de Ciências Naturais e HumanasUniversidade Federal do ABCSão PauloBrazil
  3. 3.Instituto de Física de São Carlos (IFSC)Universidade de São Paulo (USP)São CarlosBrazil
  4. 4.Institut de Biologie Structurale (IBS), Grenoble, Alpes, CEA, CNRSGrenobleFrance

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