Comprehensive profiling of translation initiation in influenza virus infected cells

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 lactidomycin 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. Host transcripts induced by the anti-viral response are enriched for translation initiation at non-canonical start sites and non-AUG start codons. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

Antiviral activity of iridoid glycosides extracted from Fructus Gardeniae against influenza A virus by PACT-dependent suppression of viral RNA replication

“…To verify the activation of PACT and eIF2α induced by IAV infection, we measured PACT expression and eIF2α phosphorylation in infected A549 cells 12, 24, 36, and 48 h post-infection. In the current study, PACT general agreement with a previous report 56 . Furthermore, IGEs treatment markedly inhibited PACT expression in a dose-dependent manner at the three measurement time points post-infection.…”

Accessory Gene Products of Influenza A Virus

“…3D). It was discovered in a PR8 reassortant virus with WSN segment 6 as an unexpected peak in a ribosome profiling (Ribo-seq) data set generated using the inhibitor lactimidomycin to bias reads toward sites of translation initiation (Machkovech et al 2019). Initiation at the second AUG present in the mRNA (codon 15 in the NA gene, starting at the 43rd nucleotide downstream of the primary NA AUG, hence the protein name chosen by the authors) seems likely to occur via leaky scanning; the primary AUG, serving as the initiation site for NA translation, is in a poor Kozak consensus and the second (NA43) AUG is in a moderate Kozak consensus (Fig.…”

“…Most, but not all, N1 NAs have this methionine codon, suggesting that NA43 expression may be widespread. The existence of the truncated NA polypeptide is well supported by plasmid-based experiments for WSN and the early H1N1pdm09 strain A/California/04/2009, but has not been unequivocally demonstrated in actual virus infection (Machkovech et al 2019). Despite the partial loss of the transmembrane domain, exogenously expressed NA43 still accumulates on the plasma membrane.…”

“…Despite the partial loss of the transmembrane domain, exogenously expressed NA43 still accumulates on the plasma membrane. However, probing its importance through reverse genetics gave no indication of functional importance for replication in vitro or in mice (Machkovech et al 2019), so its significance remains unclear.…”

“…Other evidence for noncanonical gene expression events in IAV has come from the technique of ribosomal profiling. This technique, which maps fragments of mRNA protected from RNase digestion by ribosomes back to the genome to (among other things) identify sites of translation initiation (Ingolia 2016), has identified at least four previously unrecognized ribosome start sites in the IAV genome (Machkovech et al 2019). These are internal methionine codons at positions 86 and 136 of the PA and NP genes (Fig.…”