Biochemical characterization of the respiratory syncytial virus P-P and P-N protein complexes and localization of the P protein oligomerization domain.
The RNA-dependent RNA polymerase complex of respiratory syncytial virus (RSV) is composed of the large polymerase (L), the phosphoprotein (P), the nucleocapsid protein (N) and the co-factors M2-1 and M2-2. The P protein plays a central role within the replicase-transcriptase machinery, forming homo-oligomers and complexes with N and L. In order to study P-P and N-P complexes, and the role of P phosphorylation in these interactions, the human RSV P and N proteins were expressed in E. coli as His-tagged or GST-fusion proteins. The non-phosphorylated status of recombinant P protein was established by mass spectrometry. GST-P and GST-N fusion proteins were able to interact with RSV proteins extracted from infected cells in a GST pull-down assay. When co-expressed in bacteria, GST-P and His-P were co-purified by glutathione-Sepharose affinity, showing that the RSV P protein can form oligomers within bacteria. This result was confirmed by chemical cross-linking experiments and gel filtration studies. The P oligomerization domain was investigated by a GST pull-down assay using a series of P deletion constructs. This domain was mapped to a small region situated in the central part of P (aa 120-150), which localized in a computer-predicted coiled-coil domain. When co-expressed in bacteria, RSV N and P proteins formed a soluble complex that prevented non-specific binding of N to bacterial RNA. Therefore, RSV P protein phosphorylation is not required for the formation of P-P and N-P complexes, and P controls the RNA binding activity of N.
Castagné
Nathalie
N
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Barbier
Alexandra
A
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Bernard
Julie
J
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Rezaei
Human
H
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Huet
Jean-Claude
JC
Unité de Biochimie et Structure des Protéines, INRA, 78350 Jouy-en-Josas, France.
Henry
Céline
C
Unité de Biochimie et Structure des Protéines, INRA, 78350 Jouy-en-Josas, France.
Costa
Bruno Da
BD
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
Eléouët
Jean-François
JF
Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012329:RNA, Bacterial / Q000032:analysis; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012136:Respiratory Syncytial Viruses / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*
D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins