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

Abstract

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.