title: Probing bunyavirus N protein oligomerisation using mass spectrometry.
abstract: BACKGROUND
  Bunyaviruses have become a major threat to both humans and livestock in Europe and the Americas. The nucleocapsid (N) protein of these viruses is key to the replication cycle and knowledge of the N oligomerisation state is central to understanding the viral lifecycle and for development of therapeutic strategies.
  
  
  METHODS
  Bunyamwera virus and Schmallenberg virus N proteins (BUNV-N and SBV-N) were expressed recombinantly in E. coli as hexahistidine-SUMO-tagged fusions, and the tag removed subsequently. Noncovalent nano-electrospray ionisation mass spectrometry was conducted in the presence and absence of short RNA oligonucleotides. Instrumental conditions were optimised for the transmission of intact protein complexes into the gas phase. The resulting protein-protein and protein-RNA complexes were identified and their stoichiometries verified by their mass. Collision-induced dissociation tandem mass spectrometry was used in cases of ambiguity.
  
  
  RESULTS
  Both BUNV-N and SBV-N proteins reassembled into N-RNA complexes in the presence of RNA; however, SBV-N formed a wider range of complexes with varying oligomeric states. The N:RNA oligomers observed were consistent with a model of assembly via stepwise addition of N proteins. Furthermore, upon mixing the two proteins in the presence of RNA no heteromeric complexes were observed, thus revealing insights into the specificity of oligomerisation.
  
  
  CONCLUSIONS
  Noncovalent mass spectrometry has provided the first detailed analysis of the co-populated oligomeric species formed by these important viral proteins and revealed insights into their assembly pathways. Using this technique has also enabled comparisons to be made between the two N proteins.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Shepherd
authors.forename: Antonio
authors.initials: A
authors.lastname: Ariza
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Edwards
authors.forename: John N
authors.initials: JN
authors.lastname: Barr
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Stonehouse
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
mesh_terms: D000595:Amino Acid Sequence; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: C423924:N protein, Bunyamwera bunyavirus; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins