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 (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.
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Filip
F
Yabukarski
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Cedric
C
Leyrat
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France; Institut Laue Langevin, Grenoble, France.
Nicolas
N
Martinez
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Guillaume
G
Communie
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France; Institut Laue Langevin, Grenoble, France.
Ivan
I
Ivanov
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Euripedes A
EA
Ribeiro
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France; Laboratoire de Virologie, Centre Hospitalo-Universitaire de Grenoble, Grenoble, France.
Marlyse
M
Buisson
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Francine C
FC
Gerard
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Jean-Marie
JM
Bourhis
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Malene Ringkjøbing
MR
Jensen
Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, Université Montpellier 1 and 2, Montpellier, France.
Pau
P
Bernadó
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France.
Martin
M
Blackledge
Institut de Biologie Structurale (IBS), CEA, CNRS, Université Grenoble Alpes, Grenoble 38044, France. Electronic address: marc.jamin@ibs.fr.
Marc
M
Jamin
D001665:Binding Sites; D018832:Molecular Chaperones / Q000378:metabolism*; D019251:Nucleocapsid / Q000378:metabolism; D009698:Nucleoproteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D054243:Vesicular Stomatitis / Q000821:virology; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*
D018832:Molecular Chaperones; D009698:Nucleoproteins; D010750:Phosphoproteins; D012367:RNA, Viral; D015678:Viral Structural Proteins
negative-strand RNA viruses; nucleocapsid assembly; rhabdoviruses; small-angle scattering; viral replication