Journal Article

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Aumont-Nicaise, Magali, Bontems, François, Eléouët, Jean-François, Dubosclard, Virginie, Blondot, Marie-Lise, Lassoued, Safa, Fix, Jenna, Sizun, Christina

2012

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Biology / Chemical Sciences / Ebola virus / Genes / Genetic transcription / Genomes / Inclusion Bodies, Viral / Mutagenesis, Site-Directed / Mutation / Nuclear magnetic resonance / Nuclear Magnetic Resonance, Biomolecular / Organic chemistry / Point Mutation / Protein Binding / Protein Interaction Domains and Motifs / Protein Structure, Tertiary / Proteins / Recombinant Proteins / Respiratory syncytial virus / RNA polymerase / RNA, Viral - chemistry / RNA, Viral - genetics / RNA-Binding Proteins - chemistry / RNA-Binding Proteins - genetics / Structure / Transcription factors / Transcription, Genetic / Viral Structural Proteins - chemistry / Viral Structural Proteins - genetics