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

The Respiratory syncytial virus M2-1 protein forms tetramers and interacts with RNA and P in a competitive manner

The Respiratory Syncytial Virus (RSV) M2-1 protein is an essential cofactor of the viral RNA polymerase complex and functions as a transcriptional processivity and antitermination factor. M2-1, which exists as a phosphorylated or non-phosphorylated form in infected cells, is an RNA-binding protein that also interacts with some of the other components of the viral polymerase complex. It contains a CCCH motif at the N-terminus, a putative zinc-binding domain that is essential for M2-1 function. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in E. coli and purified to > 95% homogeneity using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were co-purified with bacterial RNA that could be eliminated by high salt wash. Circular dichroism showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity and electron microscopy led to the conclusion that M2-1 forms a 5.4 S tetramer of 89 kDa and of approximately 7.6 nm in diameter at micromolar concentrations. By using a series of deletion mutants, the oligomerization domain of M2-1 was mapped to a putative alpha-helix consisting of amino acid residues 32-63. When tested in an RSV minigenome replicon system using luciferase as a reporter gene, a M2-1 deletion mutant lacking this region showed a significant reduction in RNA transcription compared to wild-type M2-1, indicating that M2-1 oligomerization is essential for its activity. We also show that the region encompassing amino acid residues 59-178 binds to P and RNA in a competitive manner that is independent of M2-1's phosphorylation status

On the Role of NMR Spectroscopy for Characterization of Antimicrobial Peptides

Antimicrobial peptides (AMPs) provide a primordial source of immunity, conferring upon eukaryotic cells resistance against bacteria, protozoa, and viruses. Despite a few examples of anionic peptides, AMPs are usually relatively short positively charged polypeptides, consisting of a dozen to about a hundred amino acids, and exhibiting amphipathic character. Despite significant differences in their primary and secondary structures, all AMPs discovered to date share the ability to interact with cellular membranes, thereby affecting bilayer stability, disrupting membrane organization, and/or forming well-defined pores. AMPs selectively target infectious agents without being susceptible to any of the common pathways by which these acquire resistance, thereby making AMPs prime candidates to provide therapeutic alternatives to conventional drugs. However, the mechanisms of AMP actions are still a matter of intense debate. The structure-function paradigm suggests that a better understanding of how AMPs elicit their biological functions could result from atomic resolution studies of peptide-lipid interactions. In contrast, more strict thermodynamic views preclude any roles for three-dimensional structures. Indeed, the design of selective AMPs based soley on structural parameters has been challenging. In this chapter, we will focus on selected AMPs for which studies on the corresponding AMP-lipid interactions have helped reach an understanding of how AMP effects are mediated. We will emphasize the roles of both liquid- and solid-state NMR spectroscopy for elucidating the mechanisms of action of AMPs