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Fine mapping and characterization of the binding domain of the HRSV Phosphoprotein with the M2-1 protein
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The RSV genome is transcribed into 10 mRNAs by the RNA-dependant RNA polymerase complex (RdRp). M2-1 protein is a transcription antiterminator which increases the processivity of the RdRp during transcription. M2-1 is recruited to RNA transcription sites by the phosphoprotein P. Since protein-protein interactions are a target for antiviral compounds, our objective is to obtain the crystallographic structure of the M2-1—P complex. The atomic structure of full-length tetrameric M2-1 is now available. However, since P is a naturally disordered protein, it is not possible to use full-length P for that purpose. The aim of this work was to finely characterise the M2-1 binding domain of P and to use this domain for co-crystallization trials. The M2-1-binding domain of P was previously mapped to residues 100-120 by internal deletions by Mason et al. By using NMR, we identified P residues ~ 90-100 as a region interacting with M2-1. Using recombinant proteins and deletions, the M2-1 binding site was finely mapped to amino acid residues 93-110. The role of amino acid residues in M2-1—P interaction was investigated by site-directed mutagenesis and pull-down assays, and the impact of these mutations on viral transcription was evaluated in cellula using an RSV minigenome. The results highlighted the critical role of some residues located in this region. The role of P oligomerization for M2-1—P interaction was also investigated.
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