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 m2-158-177 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 m2-158-177,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. © 2012 blondot et al.