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 M2-158–177 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.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

“…FA analysis of mutant R3AR4A shows that these residues also influence RNA binding, implying that M2-1 residues that participate in RNA binding are not solely confined to the basic cluster of the core. We saw no significant difference between mutating selected positively charged residues to alanine or aspartate, unlike in previous studies where introducing negative charge increased loss of RNA binding affinity relative to neutralizing positive charge (12), and this discrepancy may reflect the different methods of analysis.…”

“…These data indicate that tetrameric full-length M2-1 exhibits a preference for poly-A or A-rich sequences, particularly in the context of mRNA sense gene ends, as has been previously shown for monomeric M2-1 58-177 by NMR (12). In contrast to this previous work, however, full-length tetrameric M2-1 protein displays a considerable difference (over fivefold) in binding affinity to RNA sequences representing gene ends from different junctions.…”

“…Extensive contact of the ZBD with the adjacent monomer is consistent with its requirement for M2-1 function and implies that it is important for tetramer integrity. In the tetramer context, the ZBDs lie on the N-terminal face in close proximity to the RNA binding surface (12), raising the possibility of involvement in RNA binding (Fig. 1A).…”

“…M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11). Based on mutational analysis and a partial M2-1 structure determined using NMR (12,13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exhibits significant structural homology with the Ebola virus (EBOV) VP30 transcription factor (12), which interestingly also possesses an N-terminal zinc-binding domain (15) and phosphorylation sites that regulate VP30 function (16).…”

“…The RNA binding specificity of M2-1 is still debated, with antigenomic leader, viral mRNA, poly-A, or no sequence specificity having been proposed (9,11,12,17). In this article, we present the crystal structure of full-length, tetrameric HRSV M2-1 protein in both wild type (WT) and phosphomimetic forms.…”