Influenza A virus utiizes RNA throughout infection. Little is known, however, about the roles of RNA structures in these processes. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure is 39 nucleotides downstream of the M2 mRNA 5′ splice site. Here a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multi-branch structure of this RNA. Imino proton spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ~14 °C higher than for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture.
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