The 3′ Splice Site of Influenza A Segment 7 mRNA Can Exist in Two Conformations: A Pseudoknot and a Hairpin

Walter N Moss,Lumbini I Dela-Moss,Elzbieta Kierzek,Douglas H Turner,Ryszard Kierzek,Salvatore F Priore,Alfred Lewin

doi:10.1371/journal.pone.0038323

Walter N Moss, Lumbini I Dela-Moss + Show 5 more

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https://doi.org/10.1371/journal.pone.0038323

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Abstract

The 3′ splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3′ splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

Highlights

Pandemic outbreaks of influenza A were responsible for millions of deaths in the 20th century
A similar hairpin/pseudoknot was described for the 39 splice site of segment 8, which was proposed to influence splicing of the NS2 mRNA [16,17]. These results suggest that splicing of segment 7 may be modulated by varying splice site accessibility [18,19,20,21] or splicing factor binding [22,23,24], and that conformational switching may be a common mechanism to control splicing of influenza genes
A 39 splice site (3PSS) from segment 7, alongside an artificial mutant construct (HPMut) that can fold into a hairpin but not

Summary

Introduction

Pandemic outbreaks of influenza A were responsible for millions of deaths in the 20th century. The Spanish Flu of 1918 killed between 20 [1] and 100 million people [2]. Influenza is still of grave concern to public health. Most therapeutics target influenza proteins: e.g. blocking the M2 ion channel with amantadine and rimantadine [7]). The virus, utilizes RNA at every step in its propagation, making viral RNA an attractive target for therapeutic treatment [8,9]. A better understanding of the structure and function of RNA in influenza A would open new avenues for treatment of this deadly disease, and provide a valuable complement to current therapeutics

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