Date Published: January 23, 2019
Publisher: Public Library of Science
Author(s): Heather M. Machkovech, Jesse D. Bloom, Arvind R. Subramaniam, Jonathan W Yewdell.
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 is 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.
Cellular stress can impact protein translation initiation. For example, viral infection can activate protein kinase R, which globally reduces translation initiation for most transcripts, while translationally upregulating a subset of stress-response transcripts . Cellular stress can also alter the start codon on mRNAs at which ribosomes initiate translation [2–4]. A common alternate start codon, CUG, may be of particular relevance during immune stress. Previous work using sensitive reporter assays found that CUG initiation can be induced during viral infection and activation of the immune response [5, 6]. It has been hypothesized that upregulation of CUG initiation may be a deliberate host strategy to generate immune epitopes during cellular stress [5–7]. Indeed, there are numerous examples of immune epitopes that are initiated from alternate start sites, including CUG codons [8–13]. However, it is unknown if start site usage is altered globally during viral infection, or skewed towards certain start codons such as CUG.
We have performed the first comprehensive characterization of translation initiation sites on viral and host transcripts during influenza virus infection. In viral transcripts, we identified a total of 14 high confidence translation initiation sites, including 7 of 8 canonical translation initiation sites and two previously characterized non-canonical translation initiation sites: PB1-F2  and a start site at nucleotide 113 of M . The seven alternate start sites that we identified were distributed across the PB1, M, NP, NA, and PA segments, and we found candidate novel N-terminal truncations in NP, PA, and NA.