Tran, T. L. and Castagné, N. and Dubosclard, V. and Noinville, S. and Koch, E. and Moudjou, M. and Henry, C. and Bernard, J. and Yeo, R. P. and Eléouët, J. F. (2009) 'The respiratory syncytial virus M2-1 protein forms tetramers and interacts with RNA and P in a competitive manner.', Journal of virology., 83 (13). pp. 6363-6374.
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.
|Keywords:||RSV, M2-1, Oligomer, Interaction, Polymerase.|
|Full text:||Full text not available from this repository.|
|Publisher Web site:||http://dx.doi.org/10.1128/JVI.00335-09|
|Record Created:||19 Jun 2009 10:05|
|Last Modified:||24 Jun 2009 12:19|
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