Ensemble Structure of the Highly Flexible Complex Formed between Vesicular Stomatitis Virus Unassembled Nucleoprotein and its Phosphoprotein Chaperone

J Mol Biol. 2016 Jul 3;428(13):2671-94. doi: 10.1016/j.jmb.2016.04.010. Epub 2016 Apr 21.


Nucleocapsid assembly is an essential process in the replication of the non-segmented, negative-sense RNA viruses (NNVs). Unassembled nucleoprotein (N(0)) is maintained in an RNA-free and monomeric form by its viral chaperone, the phosphoprotein (P), forming the N(0)-P complex. Our earlier work solved the structure of vesicular stomatitis virus complex formed between an N-terminally truncated N (NΔ21) and a peptide of P (P60) encompassing the N(0)-binding site, but how the full-length P interacts with N(0) remained unknown. Here, we combine several experimental biophysical methods including size exclusion chromatography with detection by light scattering and refractometry, small-angle X-ray and neutron scattering and nuclear magnetic resonance spectroscopy with molecular dynamics simulation and computational modeling to characterize the NΔ21(0)-PFL complex formed with dimeric full-length P. We show that for multi-molecular complexes, simultaneous multiple-curve fitting using small-angle neutron scattering data collected at varying contrast levels provides additional information and can help refine structural ensembles. We demonstrate that (a) vesicular stomatitis virus PFL conserves its high flexibility within the NΔ21(0)-PFL complex and interacts with NΔ21(0) only through its N-terminal extremity; (b) each protomer of P can chaperone one N(0) client protein, leading to the formation of complexes with stoichiometries 1N:P2 and 2N:P2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N(0) but creates a metal binding site in PNTR. A comparison with the structures of Nipah virus and Ebola virus N(0)-P core complex suggests a mechanism for the control of nucleocapsid assembly that is common to all NNVs.

Keywords: negative-strand RNA viruses; nucleocapsid assembly; rhabdoviruses; small-angle scattering; viral replication.

MeSH terms

  • Binding Sites
  • Molecular Chaperones / metabolism*
  • Nucleocapsid / metabolism
  • Nucleoproteins / metabolism*
  • Phosphoproteins / metabolism*
  • Protein Binding / genetics
  • RNA, Viral / genetics
  • Vesicular Stomatitis / virology
  • Vesicular stomatitis Indiana virus / metabolism*
  • Viral Structural Proteins / metabolism*


  • Molecular Chaperones
  • Nucleoproteins
  • Phosphoproteins
  • RNA, Viral
  • Viral Structural Proteins