title: 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 in a phosphorylated or unphosphorylated 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, a putative zinc-binding domain that is essential for M2-1 function, at the N terminus. To gain insight into its structural organization, M2-1 was produced as a recombinant protein in Escherichia coli and purified to >95% homogeneity by using a glutathione S-transferase (GST) tag. The GST-M2-1 fusion proteins were copurified with bacterial RNA, which could be eliminated by a high-salt wash. Circular dichroism analysis showed that M2-1 is largely alpha-helical. Chemical cross-linking, dynamic light scattering, sedimentation velocity, and electron microscopy analyses led to the conclusion that M2-1 forms a 5.4S tetramer of 89 kDa and 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 to 63. When tested in an RSV minigenome replicon system using a luciferase gene as a reporter, an 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 the activity of the protein. We also show that the region encompassing amino acid residues 59 to 178 binds to P and RNA in a competitive manner that is independent of the phosphorylation status of M2-1.
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France.
authors.forename: Thi-Lan
authors.initials: TL
authors.lastname: Tran
authors.forename: Nathalie
authors.initials: N
authors.lastname: Castagné
authors.forename: Virginie
authors.initials: V
authors.lastname: Dubosclard
authors.forename: Sylvie
authors.initials: S
authors.lastname: Noinville
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Koch
authors.forename: Mohammed
authors.initials: M
authors.lastname: Moudjou
authors.forename: Céline
authors.initials: C
authors.lastname: Henry
authors.forename: Julie
authors.initials: J
authors.lastname: Bernard
authors.forename: Robert Paul
authors.initials: RP
authors.lastname: Yeo
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eléouët
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000378:metabolism*
chemical_list: C529603:P protein, human respiratory syncytial virus; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins