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

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 (N-Delta 21) and a peptide of P (P-60) 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 N-Delta 21(0)-P-FL 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 P-FL conserves its high flexibility within the N-Delta 21(0)-P-FL complex and interacts with N-Delta 21(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:P-2 and 2N:P-2; and (c) phosphorylation of residues Ser60, Thr62 and Ser64 provides no additional interactions with N-0 but creates a metal binding site in P-NTR. 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. (C) 2016 Elsevier Ltd. All rights reserved.