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. 2014 Apr 15;28(7):793-800.
doi: 10.1002/rcm.6841.

Probing bunyavirus N protein oligomerisation using mass spectrometry

Dale A Shepherd  1 , Antonio Ariza, Thomas A Edwards, John N Barr, Nicola J Stonehouse, Alison E Ashcroft
Affiliations

Probing bunyavirus N protein oligomerisation using mass spectrometry

Dale A Shepherd et al. Rapid Commun Mass Spectrom. .

Abstract

Rationale: 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.

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Figures

Figure 1
Figure 1
The crystal structures of BUNV-N and SBV-N. (a) Overlay of BUNV-N (yellow, PDB ID 3ZLA) and SBV-N (maroon, PDB ID 3ZL9) monomer crystal structures taken from the crystallographic tetramers, illustrating similarities in the fold; (b) BUNV-N tetramer bound to RNA (orange stripe); (c) sequence alignment of the N proteins of BUNV, SBV, LACV, and LEAV. Degree of conservation (percentage identity) is indicated in shades of blue.
Figure 2
Figure 2
ESI-MS shows that RNA is crucial for N protein complex stability. (a) BUNV-N with endogenous E. coli RNA. The observed complex (20+ to 26+ charge state ions) has a mass of 120 894 Da consistent with a BUNV-N tetramer bound to 44 nt of RNA; (b) BUNV-N is predominantly monomeric (mass 26 751 Da; 8+ to 11+ charge state ions, green triangles) after removal of the endogenous RNA. Inset: expansion of m/z 5000–7000 region showing traces of RNA-bound tetramers and pentamers remaining after the RNA-removal step (red and navy triangles, respectively); (c) SBV-N exists as a monomer (mass 26 269 Da) (8+ to 10+ charge state ions, green circles), tetramer (m/z 5000–6000, red circles), and pentamer (m/z 5500–7000, navy circles) in the absence of endogenous RNA.
Figure 3
Figure 3
ESI-MS analysis of BUNV-N with added synthetic RNAs. (a) BUNV-N in the presence of the 12-mer RNA forms complexes of [(BUNV)3 + (12mer)2] (purple triangles), [(BUNV)3 + (12mer)3] (blue triangles), and [(BUNV)4 + (12-mer)4] (red triangles); (b) BUNV-N in the presence of the 24-mer RNA forms complexes of [(BUNV)3 + (24-mer)] (blue triangles), [(BUNV)4 + (24-mer)2] (red triangles), [(BUNV)5 + (24-mer)2] (navy triangles), and [(BUNV)6 + (24-mer)3] (grey triangles); (c) BUNV-N in the presence of the 48-mer RNA forms complexes of [(BUNV)5 + (48-mer)]; inset: expansion of m/z 4500–8000 region; (d) BUNV-N in the presence of the 60-mer RNA forms complexes of [(BUNV)6 + (60-mer)]; inset: expansion of m/z 4500–8000 region. Under both the 48 nt and 60 nt conditions there is a contribution from E. coli RNA-bound N (grey signals).
Figure 4
Figure 4
ESI-MS analysis of SBV-N with synthetic RNAs. (a) SBV-N in the presence of the 12-mer RNA forms complexes of [(SBV)4 + (12-mer)4] (red circles), [(SBV)8 + (12-mer)8] (yellow circles), [(SBV)10 + (12-mer)10] (green circles), and [(SBV)12 + (12-mer)12] (purple circles); (b) SBV-N in the presence of the 24-mer RNA forms complexes of [(SBV)4 + (24-mer)2] (red circles) and [(SBV)5 + (24-mer)2] (navy circles); (c) SBV-N in the presence of the 48-mer RNA forms complexes of [(SBV)3 + (48-mer)] (purple circles) and [(SBV)4 + (48-mer)] (red circles) as well as small amounts of [(SBV)4 + (48-mer)2] (blue circles) and [(SBV)5 + (48-mer)2] (beige circles); (d) SBV-N in the presence of the 60-mer RNA forms complexes of [(SBV)4 + (60-mer)] (red circles), [(SBV)5 + (60-mer)] (navy circles), [(SBV)6 + (60mer)] (grey circles), and [(SBV)8 + (60-mer)] (yellow circles).
Figure 5
Figure 5
ESI-MS shows that oligomerisation is cooperative between RNA and protein. (a) RNA-free SBV-N tetramers (red circles) and pentamers (navy circles) are not disrupted by possible competing interactions with BUNV-N (monomers, green triangles), suggesting incompatible N-N interfaces, inset: expansion of m/z 4500–7000 region; (b) 1:1 molar BUNV-N: SBV-N in the presence of the 24-mer RNA (red squares, 2:1 total protein:RNA). The resulting RNA-protein complexes are identified as [(BUNV)4 + (24-mer)2] (red triangles) and [(SBV)4 + (24-mer)2] (red circles), inset: expansion of m/z 4500–7000 region; (c) CID MS/MS of the 20+ charge state ions of [(SBV)4 + (24-mer)2] (red circles). Only the expected SBV-N monomer (11+ to 13+) (green circles) and trimeric stripped complex ions (7+ to 9+) (blue circles) are observed; (d) CID MS/MS of the 22+ charge state ions of [(BUNV)4 + (24-mer)2] (red triangles). Only the expected BUNV-N monomer (12+ to 14+) (green triangles) and stripped trimeric complex ions (8+ to 12+) (blue triangles) are observed. Thus, the MS/MS data verify the MS data in (b).
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