Identification of cis-Acting Elements that Regulate Influenza A Virus Segment 7 Differential mRNA Splicing

Abstract

Influenza A virus segment 7 encodes the M1 matrix protein and M2 ion channel from an unspliced primary transcript (mRNA1) and spliced mRNA2, respectively. Both proteins are essential for efficient virus replication, but the intrinsic sequences that determine segment 7 splicing balance are not fully defined. Here, we systematically investigated how conserved cis-acting splicing elements regulate segment splicing. We found that while the splice acceptor sequence is optimal, both potential upstream polypyrimidine tracts regulate splicing, with the 5' region acting positively and the 3' region negatively to maintain M1 expression. Nucleotide A713 is the most likely mRNA2 branch point, but potentially with redundancy from other local adenosines. Mutagenesis of the mRNA2 splice donor sequence showed that it is nearly optimal but that nucleotides 51 and 55 modestly downregulate splicing efficiency. Most introduced mutations that change splicing balance are nonsynonymous in M1. Nevertheless, polypyrimidine tract and branch point mutations that significantly perturbed M1:M2 balance resulted in viable viruses. In contrast, the splice donor sequence was largely intolerant of mutation. We conclude that M1:M2 balance is maintained via suboptimal splice donor and polypyrimidine tract splicing signals, with the latter allowing more evolutionary flexibility to vary the ratio and/or M1 sequence