Comprehensive profiling of translation initiation in influenza virus infected cells

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response are enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape. Author summary When viruses such as influenza infect cells, both host and viral mRNAs are translated into proteins. Here we investigate the sites in these mRNAs that initiate protein translation during influenza infection. In particular, we explore whether some of this translation initiates at codons other than the canonical ones used to produce the primary protein product of each gene. Using computational analyses, we find that mammalian influenza viruses evolve to reduce the number of codons that can initiate such alternate translation initiation products. We next use the comprehensive experimental strategy of ribosome profiling to identify sites of translation initiation across all influenza and host mRNAs. We find a number of sites of alternate initiation on both influenza and host mRNAs. We study in detail one such alternate start site on an influenza mRNA, and show that it encodes a functional and previously uncharacterized variant of a viral protein. We also find evidence that the mRNAs that host cells express at higher levels during viral infection are enriched for translation initiation at non-canonical start codons. Overall, these results suggest that alternate translation initiation plays a role in shaping the repertoires of both viral and host proteins that are produced during influenza infection.