Investigating the evolutionary plasticity of influenza virus splicing using influenza A virus segment 7 as a model system

Abstract

Influenza viruses A, C and D (IAV, ICV, IDV respectively) utilise mRNA splicing to produce two proteins from their matrix gene segment; the matrix (M1) protein and an ion channel (M2). In IAV, the M1 and M2 open reading frames (ORFs) partially overlap and splice donor (SD) and splice acceptor (SA) sites in the M1 gene are used to produce an M2 polypeptide with an N-terminal sequence shared with M1. In ICV and IDV, the M1 and M2 ORFs are concatenated and splice sites downstream of the M1 coding region are used to produce a matrix protein from the spliced mRNA. To probe how these different splicing strategies might have evolved, we used reverse genetics to test the evolutionary flexibility of IAV SD and SA sites. We found that relatively small numbers of mutations were sufficient to activate a variety of alternative SD and SA sites in a mini-replicon system, several of which led to the production of ion channel isoforms with variant ectodomains, as well as one that produced an M1 variant. Furthermore, several of the viruses with altered splicing patterns were viable, including five with both SD and SA sites changed. Three of these 'rewired' viruses had only modest fitness penalties, despite up to 3 nonsynonymous changes in M1 and a 34 amino acid insertion in M2. These results suggest that IAV segment 7 splicing strategies are evolutionarily flexible and have implications for how IAV, ICV and IDV matrix segments might have diversified from a common ancestor.