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Journal of Virology, July 2009, p. 6363-6374, Vol. 83, No. 13
0022-538X/09/$08.00+0     doi:10.1128/JVI.00335-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Respiratory Syncytial Virus M2-1 Protein Forms Tetramers and Interacts with RNA and P in a Competitive Manner{triangledown}

Thi-Lan Tran,1 Nathalie Castagné,1 Virginie Dubosclard,1 Sylvie Noinville,1 Emmanuelle Koch,1 Mohammed Moudjou,1 Céline Henry,2 Julie Bernard,1 Robert Paul Yeo,3 and Jean-François Eléouët1*

INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France,1 INRA, Unité Biochimie et Structure des Protéines UR477, F-78350 Jouy-en-Josas, France,2 Centre for Bioactive Chemistry, Department of Chemistry, University Science Laboratories, South Road, Durham DH1 3LE, United Kingdom3

Received 14 February 2009/ Accepted 17 April 2009

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 ~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.

* Corresponding author. Mailing address: INRA, Unité de Virologie Immunologie Moléculaires UR892, F-78350 Jouy-en-Josas, France. Phone: (33) 1 34 65 26 04. Fax: (33) 1 34 65 26 21. E-mail: jean-francois.eleouet{at}jouy.inra.fr

{triangledown} Published ahead of print on 22 April 2009.

Journal of Virology, July 2009, p. 6363-6374, Vol. 83, No. 13
0022-538X/09/$08.00+0     doi:10.1128/JVI.00335-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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