Advertisement

Amino Acids

, Volume 50, Issue 6, pp 711–721 | 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

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.

Keywords

Oropouche Viral protein Nucleoprotein–RNA complex Arboviruses 

Notes

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.

Funding

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).

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)

References

  1. Afonso A, Conraths F (2014) Schmallenberg virus preventive veterinary. Medicine 116:337–338.  https://doi.org/10.1016/j.prevetmed.2014.09.007 Google Scholar
  2. Ariza A et al (2013) Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization. Nucl Acid Res 41:5912–5926.  https://doi.org/10.1093/nar/gkt268 CrossRefGoogle Scholar
  3. Baklouti A et al (2017) Toscana virus nucleoprotein oligomer organization observed in solution Acta crystallographica Section D. Struct Biol 73:650–659.  https://doi.org/10.1107/S2059798317008774 Google Scholar
  4. Balfour HH Jr, Edelman CK, Bauer H, Siem RA (1976) California arbovirus (La Crosse) infections. III. Epidemiology of California encephalitis in Minnesota. J Infect Di 133:293–301CrossRefGoogle Scholar
  5. Bastos Mde S et al (2012) Identification of Oropouche Orthobunyavirus in the cerebrospinal fluid of three patients in the Amazonas, Brazil. Am J Trop Med Hyg 86:732–735.  https://doi.org/10.4269/ajtmh.2012.11-0485 CrossRefPubMedGoogle Scholar
  6. Beaty BJ, Calisher CH (1991) Bunyaviridae–natural history. Curr Top Microbiol Immunol 169:27–78PubMedGoogle Scholar
  7. 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
  8. Bowden TA, Bitto D, McLees A, Yeromonahos C, Elliott RM, Huiskonen JT (2013) Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike. PLoS Pathog 9:e1003374.  https://doi.org/10.1371/journal.ppat.1003374 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Broce S et al (2016) Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals. Biol Direct 11:25.  https://doi.org/10.1186/s13062-016-0126-5 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cardoso BF et al (2015) Detection of Oropouche virus segment S in patients and inCulex quinquefasciatus in the state of Mato Grosso. Br Mem Inst Oswaldo Cruz 110:745–754.  https://doi.org/10.1590/0074-02760150123 CrossRefGoogle Scholar
  11. Chen VB et al (2010) MolProbity: all-atom structure validation for macromolecular crystallography acta crystallographica Section D. Biol Crystallogr 66:12–21.  https://doi.org/10.1107/S0907444909042073 CrossRefGoogle Scholar
  12. Chowdhary R et al (2012) Genetic characterization of the Wyeomyia group of orthobunyaviruses and their phylogenetic relationships. J Gen Virol 93:1023–1034.  https://doi.org/10.1099/vir.0.039479-0 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dong H, Li P, Bottcher B, Elliott RM, Dong C (2013a) Crystal structure of Schmallenberg orthobunyavirus nucleoprotein-RNA complex reveals a novel RNA sequestration mechanism. RNA 19:1129–1136.  https://doi.org/10.1261/rna.039057.113 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Dong H, Li P, Elliott RM, Dong C (2013b) Structure of Schmallenberg orthobunyavirus nucleoprotein suggests a novel mechanism of genome encapsidation. J Virol 87:5593–5601.  https://doi.org/10.1128/JVI.00223-13 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Duchin JS et al (1994) Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. The Hantavirus Study Group. New Engl J Med 330:949–955.  https://doi.org/10.1056/NEJM199404073301401 CrossRefPubMedGoogle Scholar
  16. Edgar RC (2004a) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinf 5:113.  https://doi.org/10.1186/1471-2105-5-113 CrossRefGoogle Scholar
  17. Edgar RC (2004b) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797.  https://doi.org/10.1093/nar/gkh340 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Eifan S, Schnettler E, Dietrich I, Kohl A, Blomstrom AL (2013) Non-structural proteins of arthropod-borne bunyaviruses: roles and functions. Viruses 5:2447–2468.  https://doi.org/10.3390/v5102447 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Elliott RM (2014) Orthobunyaviruses: recent genetic and structural insights. Nat Rev Microbiol 12:673–685.  https://doi.org/10.1038/nrmicro3332 CrossRefPubMedGoogle Scholar
  20. Ferron F et al (2011) The hexamer structure of Rift Valley fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathog 7:e1002030.  https://doi.org/10.1371/journal.ppat.1002030 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Gauci PJ et al (2015) Genomic characterisation of three Mapputta group viruses, a serogroup of Australian and Papua New Guinean bunyaviruses associated with human disease. PLoS ONE 10:e0116561.  https://doi.org/10.1371/journal.pone.0116561 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Guo Y et al (2012) Crimean-Congo hemorrhagic fever virus nucleoprotein reveals endonuclease activity in bunyaviruses. Proc Natl Acad Sci USA 109:5046–5051.  https://doi.org/10.1073/pnas.1200808109 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Guu TS, Zheng W, Tao YJ (2012) Bunyavirus: structure and replication. Adv Exp Med Biol 726:245–266.  https://doi.org/10.1007/978-1-4614-0980-9_11 CrossRefPubMedGoogle Scholar
  24. 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
  25. Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202.  https://doi.org/10.1006/jmbi.1999.3091 CrossRefPubMedGoogle Scholar
  26. Ladner JT et al (2014) Genomic and phylogenetic characterization of viruses included in the Manzanilla and Oropouche species complexes of the genus Orthobunyavirus, family Bunyaviridae. J Gen Virol 95:1055–1066.  https://doi.org/10.1099/vir.0.061309-0 CrossRefPubMedGoogle Scholar
  27. LeDuc JW, Hoch AL, Pinheiro FP, da Rosa AP (1981) Epidemic Oropouche virus disease in northern Brazil. Bull Pan Am Health Organ 15:97–103PubMedGoogle Scholar
  28. Li B et al (2013) Bunyamwera virus possesses a distinct nucleocapsid protein to facilitate genome encapsidation. Proc Natl Acad Sci USA 110:9048–9053.  https://doi.org/10.1073/pnas.1222552110 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128:82–97.  https://doi.org/10.1006/jsbi.1999.4174 CrossRefPubMedGoogle Scholar
  30. Mercer DR, Castillo-Pizango MJ (2005) Changes in relative species compositions of biting midges (Diptera: Ceratopogonidae) and an outbreak of Oropouche virus in Iquitos. Peru J Med Entomol 42:554–558CrossRefPubMedGoogle Scholar
  31. Messina JP, Pigott DM, Duda KA, Brownstein JS, Myers MF, George DB, Hay SI (2015) A global compendium of human Crimean-Congo haemorrhagic fever virus occurrence. Sci Data 2:150016.  https://doi.org/10.1038/sdata.2015.16 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Mindell JA, Grigorieff N (2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142:334–347CrossRefPubMedGoogle Scholar
  33. Mir MA, Panganiban AT (2006) The bunyavirus nucleocapsid protein is an RNA chaperone: possible roles in viral RNA panhandle formation and genome replication. RNA 12:272–282.  https://doi.org/10.1261/rna.2101906 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Niu F et al (2013) Structure of the Leanyer orthobunyavirus nucleoprotein-RNA complex reveals unique architecture for RNA encapsidation. Proc Natl Acad Sci USA 110:9054–9059.  https://doi.org/10.1073/pnas.1300035110 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Olal D et al (2014) Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein. Nucl Acids Res 42:6025–6037.  https://doi.org/10.1093/nar/gku229 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Pepin M, Bouloy M, Bird BH, Kemp A, Paweska J (2010) Rift Valley fever virus(Bunyaviridae: Phlebovirus): an update on pathogenesis, molecular epidemiology, vectors, diagnostics and prevention. Vet Res 41:61CrossRefPubMedPubMedCentralGoogle Scholar
  37. Pinheiro FP et al (1981) Oropouche virus. I. A review of clinical, epidemiological, and ecological findings. Am J Tropical Med Hygiene 30:149–160CrossRefGoogle Scholar
  38. Pinheiro FP, da Rosa APT, Gomes ML, LeDuc JW, Hoch AL (1982) Transmission of Oropouche virus from man to hamster by the midge Culicoides paraensis. Science 215:1251–1253CrossRefPubMedGoogle Scholar
  39. Qi X et al (2010) Cap binding and immune evasion revealed by Lassa nucleoprotein structure. Nature 468:779–783.  https://doi.org/10.1038/nature09605 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Raymond DD, Piper ME, Gerrard SR, Smith JL (2010) Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation. Proc Natl Acad Sci USA 107:11769–11774.  https://doi.org/10.1073/pnas.1001760107 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Reguera J, Malet H, Weber F, Cusack S (2013) Structural basis for encapsidation of genomic RNA by La Crosse Orthobunyavirus nucleoprotein. Proc Natl Acad Sci USA 110:7246–7251.  https://doi.org/10.1073/pnas.1302298110 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234:779–815.  https://doi.org/10.1006/jmbi.1993.1626 CrossRefPubMedGoogle Scholar
  43. Schober HR, Peng HL (2016) Heterogeneous diffusion, viscosity, and the Stokes-Einstein relation in binary liquids. Phys Rev E 93:052607.  https://doi.org/10.1103/physreve.93.052607 CrossRefPubMedGoogle Scholar
  44. Shepherd DA, Ariza A, Edwards TA, Barr JN, Stonehouse NJ, Ashcroft AE (2014) Probing bunyavirus N protein oligomerisation using mass spectrometry. Rapid Commun Mass Spectrometr RCM 28:793–800.  https://doi.org/10.1002/rcm.6841 CrossRefGoogle Scholar
  45. 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
  46. Svergun D, Barberato C, Koch MHJ (1995) Crysol—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystalogr 28:6CrossRefGoogle Scholar
  47. Tilston-Lunel NL, Shi X, Elliott RM, Acrani GO (2017) The potential for reassortment between oropouche and schmallenberg orthobunyaviruses. Viruses 9:2.  https://doi.org/10.3390/v9080220 CrossRefGoogle Scholar
  48. 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
  49. van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M (1996) A new generation of the IMAGIC image processing system. J Struct Biol 116:17–24.  https://doi.org/10.1006/jsbi.1996.0004 CrossRefPubMedGoogle Scholar
  50. Vasconcelos HB et al (2011) Molecular epidemiology of oropouche virus. Br Emerg Infect Dis 17:800–806.  https://doi.org/10.3201/eid1705.101333 CrossRefGoogle Scholar
  51. Wernike K et al (2015) A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences. Vet Res 46:27.  https://doi.org/10.1186/s13567-015-0165-4 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 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
  53. Wilson ML (1994) Rift Valley fever virus ecology and the epidemiology of disease emergence. Ann N Y Acad Sci 740:169–180CrossRefPubMedGoogle Scholar
  54. Xu F, Chen H, Travassos da Rosa AP, Tesh RB, Xiao SY (2007) Phylogenetic relationships among sandfly fever group viruses (Phlebovirus: Bunyaviridae) based on the small genome segment. J Gen Virol 88:2312–2319.  https://doi.org/10.1099/vir.0.82860-0 CrossRefPubMedGoogle Scholar
  55. 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
  56. Zhou H, Sun Y, Guo Y, Lou Z (2013) Structural perspective on the formation of ribonucleoprotein complex in negative-sense single-stranded RNA viruses. Trends Microbiol 21:475–484.  https://doi.org/10.1016/j.tim.2013.07.006 CrossRefPubMedGoogle Scholar

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

Personalised recommendations