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Title: Comprehensive profiling of translation initiation in influenza virus infected cells}
Article:
Abstract:
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.
Introduction:
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[1]. 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. In addition, viruses often rely on alternate translation initiation to increase the diversity of proteins encoded in their compact genomes. Influenza is an eight segmented negative stranded RNA virus that encodes ten canonical proteins, but additional influenza proteins can be generated through alternate initiation. For instance, the PB1-F2 protein is initiated at a downstream AUG in the +1 reading frame of the PB1 segment, and is thought to modulate the host immune response[14–16]. Additional peptides generated by alternate downstream AUG codons have been noted in the PB1, PA, and M influenza segments[14, 15, 17–21], but their abundance and functional relevance is unclear. There is some experimental support for CUG initiation in the non-coding region of M1 in the influenza genome[22], and very low levels of initiation on the negative sense RNA of the NS segment[23–25]. Even when such noncanonical peptides are generated at low levels and not functionally important for the virus, they can still be targeted by the host immune response[11]. However, despite the ad hoc identification of the aforementioned alternate protein products, there have been no systematic studies to map sites of translation initiation in all influenza transcripts. Ribosome profiling, the high-throughput sequencing of ribosome-protected mRNA fragments, enables genome-wide interrogation of translation[26]. Notably, ribosome profiling, in combination with inhibitors of translation initiation, can be used to identify candidate sites of translation initiation[27, 28], and unannotated proteins in viruses such as CMV[29]. Previous ribosome profiling studies during influenza virus infection have examined changes that occur at the translation level on host transcripts. Kinori et al.[30] used ribosome profiling to examine changes in translational efficiency during influenza virus infection, and concluded that viral transcripts are not preferentially translated over host transcripts. Razooki et al.[31] detected ribosome density on host lincRNAs and attributed this to translation of short, conserved open reading frames. Critically, neither of these studies attempted to comprehensively identify sites of translation initiation during influenza virus infection by applying translation initiation inhibitors. Here we integrate computational and experimental approaches to systematically examine translation initiation in influenza-infected cells. We first perform computational analyses that suggest there is selection against putative CUG alternate translation initiation sites in mammalian influenza virus lineages. We then use ribosome profiling to experimentally map sites of translation initiation in viral and cellular mRNAs during influenza virus infection. Surprisingly, we find little signal for CUG initiation in viral genes, but we do identify a number of candidate AUG start sites that are downstream of the canonical start codon. We show that one of these candidate start sites leads to production of a truncated version of the viral neuraminidase protein that is expressed on the cell surface and is enzymatically active. On the host side, we identify an excess of non-AUG start codons on transcripts that are upregulated during the anti-viral response. Together, our results highlight how alternate translation initiation can alter the protein-coding capacity of both viruses and the cells that they infect.
Discussion:
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[14] and a start site at nucleotide 113 of M[21]. 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. We biochemically validated one of the new viral alternate start sites, a downstream AUG at codon 43 of NA. We showed that the NA43 protein initiated by this start site is produced even in the context of the canonical start site, is expressed on the cell surface, and is enzymatically active. In addition, this NA43 protein is capable of supporting low levels of viral growth even in the absence of full length NA. The AUG codon at 43 is conserved in most N1 viral lineages. However, we were unable to find any evidence that NA43 impacts viral fitness in cell culture or mice, at least at the relatively low resolution with which viral fitness can be measured in laboratory settings. The N-terminal cytoplasmic tail of NA helps localize the protein to areas of viral budding on the cell surface[101–106]. Therefore, the N-terminally truncated NA43 could localize to different regions of the cell surface than full-length NA. It is interesting to speculate whether such altered localization of the NA43 protein could have some phenotypic significance, such as reducing viral coinfection[110]. One of the motivations for our study was to search for evidence of alternate translation initiation at CUG codons in influenza virus. We hypothesized that initiation at such codons might lead to immune recognition of influenza virus, since CUG codons can initiate immune epitopes[8–13], and initiation at CUG codons is thought to be upregulated during conditions of cellular stress including viral infection[5–7]. We found evolutionary signatures in the viral genome that were consistent with selection against CUG-mediated translation initiation in lineages of influenza that have adapted to mammalian hosts. However, we found minimal experiment support for CUG initiation on influenza transcripts in our ribosome profiling experiments. One possibility is that CUG initiation does generate evolutionarily relevant immune epitopes, but that the levels of CUG initiation are too low to be detected by our experimental design. For instance, CUG initiation could be partially refractory to capture by the translation initiation inhibitor lactimidomycin used in our experiments[5], or perhaps only occurs in certain types of cells. In addition, even extremely low levels of translation initiation that are difficult to detect by most experimental methods can generate peptides that can still be recognized by T-cells[25]. A second possibility is that the selection against CUG codons is due to some pressure unrelated to translation initiation. There are other evolutionary signatures of influenza adaptation to mammalian hosts with uncertain origin, such as the decrease in GC content of influenza genome during viral adaptation to mammalian hosts[42]. We also did not find significant shifts in global start site usage towards non-AUG translation initiation during either viral infection of interferon treatment, despite the fact that CUG initiation has been shown to be enhanced in these conditions using biochemical and immunological assays[6]. However, the subset of transcriptionally induced genes upon interferon-β treatment did have a significantly higher number of non-canonical translation initiation sites than other genes. Whether these alternate start sites serve any biological function will require further study. One interesting possibility is that the peptides generated from these non-canonical start sites on anti-viral genes could harbor T-cell epitopes that are relevant during the host immune response. Our ribosome profiling experiments used the translation initiation inhibitor lactimidomycin to identify candidate start sites in influenza and host transcripts. One potential concern is that the extended lactimidomycin arrest of initiating ribosomes could cause a traffic jam of scanning pre-initiation complexes[111] and lead to promiscuous initiation. Indeed more stringent initiation profiling methods have been developed to address this concern[83]. However, our goal was to detect start sites that might have very low levels of initiation. Hence we focused on achieving the highest possible sensitivity in our assay at the cost of possibly inflating the number of upstream start sites. Further, all the novel start sites identified on influenza transcripts occur downstream of the canonical initiation codon, indicating that traffic jams of pre-initiation complexes is not a major problem in analysis of the viral data. If anything, downstream start sites might be more prone to being missed by our method due to blocks caused by the initiating ribosome at the canonical start codon. Overall, our work provides the first comprehensive analysis of translation initiation during influenza virus infection. We identified several new alternate translation initiation sites, one of which produces a functionally active viral protein that has not been previously described. However, we found little evidence for large-scale initiation of translation at non-canonical start codons such as CUGs on viral transcripts, and only a modest increase in the proportion of non-canonical and start codons on host transcripts upregulated during the anti-viral response. The relative paucity of evidence for virally induced alternate translation initiation in our ribosome profiling experiments is seemingly at odds with many studies[5–13] highlighting its potential role in generating immune epitopes during viral infection, as well as our own evolutionary analyses suggesting evolutionary selection against putative CUG start sites in the viral genome. Further work will be needed to resolve this apparent conundrum and define the extent and significance of alternate translation initiation during viral infection and the resulting immune response.
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 .