Structure and functional analysis of the RNA- and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein.
Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1(58-177) core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1(58-177), as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1.
Unité de Virologie et Immunologie Moléculaires (UR892), INRA, Jouy-en-Josas, France.
Marie-Lise
ML
Blondot
Virginie
V
Dubosclard
Jenna
J
Fix
Safa
S
Lassoued
Magali
M
Aumont-Nicaise
François
F
Bontems
Jean-François
JF
Eléouët
Christina
C
Sizun
D007181:Inclusion Bodies, Viral; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D014158:Transcription, Genetic; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000235:genetics
C529603:P protein, human respiratory syncytial virus; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins