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

Abstract

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.

Author Summary

Premature termination of transcription by the RNA-dependent RNA polymerase (RdRp) complex of respiratory syncytial virus (RSV) is prevented by the M2-1 protein. This transcription factor interacts with both RNA and viral phosphoprotein P, the main RdRp cofactor, through a specific “core” domain. Using NMR, we solved the 3D structure of this domain and characterized the surface residues involved in P- and RNA-binding. Based on these data, we designed point mutations impairing binding to either RNA or P. We studied the functional implications of these mutations for transcription and co-localization of full-length M2-1 in cytoplasmic inclusion bodies, where viral transcription likely occurs. We found that the RNA- and P-binding surfaces are in close proximity, accounting for competitive binding in vitro. Our results suggest that binding to both RNA and to P is necessary for transcriptional antitermination by M2-1, but that the role of the interaction with P is primarily to recruit M2-1 to the RdRp complex. Finally we show that the M2-1 core domain is homologous to the C-terminal domain of Ebola virus VP30 despite low sequence identity, solidifying the relationship between these two proteins and transcriptional regulation strategies shared by viruses belonging to the Filoviridae family and the Pneumovirinae subfamily.