Pandemic outbreaks of influenza A were responsible for millions of deaths in the 20th
century. Notably, the Spanish Flu of 1918 killed between 20 
and 100 million people 
. Influenza is still of grave concern to public health. Each year globally there are an estimated three to five million severe infections with up to 500,000 deaths 
; in the U.S. alone there are approximately 200,000 hospitalizations and 36,000 deaths yearly 
. Most therapeutics target influenza proteins: e.g. blocking the M2 ion channel with amantadine and rimantadine 
). The virus, however, utilizes RNA at every step in its propagation, making viral RNA an attractive target for therapeutic treatment 
. 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.
The influenza A virus possesses an eight segment (–) sense RNA genome, which codes for at least eleven proteins. Fragments of the influenza A coding RNA are predicted to have unusual thermodynamic stability, and also have suppressed third codon position variability. In combination with conserved base pairing, these results provided predictions of fragments likely to fold into functional structures 
. One particularly interesting fragment () includes the 3′ splice site of segment 7, as well as key residues of a binding site for the human SF2/ASF splicing factor 
and a polypyrimidine tract that may bind other splicing factors such as U2AF65 
. Segment 7 encodes the M1 matrix protein and three alternatively spliced products that share the 3′ splice site: the M2 protein, the small M3 polypeptide, and occasionally M4 
. Production of M2 is critical for uncoating of the viral genome and splicing of the M2 mRNA is temporally controlled 
Location and structure of the 3′ splice site.
Secondary structure modeling of the 3′ splice site of segment 7 () yielded the possibility of two alternative conformations: (1) a pseudoknot (), where the splice site is base paired in a helix and (2) a hairpin (), where the splice site occurs in a two-by-two nt internal loop 
. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies reported here for a 63 nt fragment are consistent with these models. A similar hairpin/pseudoknot was described for the 3′ splice site of segment 8, which was proposed to influence splicing of the NS2 mRNA 
. These results suggest that splicing of segment 7 may be modulated by varying splice site accessibility 
or splicing factor binding 
, and that conformational switching may be a common mechanism to control splicing of influenza genes. Small molecules 
or oligonucleotides 
that specifically bind to these structures could be used to test their function and potentially provide leads for therapeutics.