The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2′, which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target.

The influenza virus NS1 protein inhibits the splicing of the major class of mammalian pre-mRNAs (GU-AG introns) by binding to a specific stem-bulge in U6 snRNA, thereby blocking the formation of U4/U6 and U2/U6 complexes. The splicing of the minor class of AT-AC introns takes place on spliceosomes that do not contain U6 snRNA, but rather U6atac snRNA — a highly divergent U6 snRNA counterpart. Nonetheless, we demonstrate that the NS1 protein inhibits AT-AC splicing in vitro, and specifically binds to only U6atac snRNA among the five minor class snRNAs. Chemical modification/interference assays show that the NS1 protein binds to the stem-bulge near the 3′ end of U6atac snRNA, encompassing nt 82–95 and nt 105–114. Although the sequence of this stem-bulge differs significantly from the sequence of the stem-bulge to which the NS1 protein binds in U6 snRNA, RNA competition experiments indicate that U6 and U6atac snRNAs likely share the same binding site on the NS1 protein. Previously, the region of U6atac snRNA containing this 3′ stem-bulge had not been implicated in any interactions of this snRNA with either U4atac or U12 snRNA. However, as assayed by psoralen crosslinking, we show that the NS1 protein inhibits the formation of U12/U6atac complexes, but not the formation of U4atac/U6atac complexes. We can conclude that the inhibition of AT-AC splicing results largely from the inhibition of formation of U12/U6atac complexes caused by the binding of the NS1 protein to the 3′ stem-bulge of U6atac snRNA.