Defining Viral Defective Ribosomal Products: Standard and Alternative Translation Initiation Events Generate a Common Peptide from Influenza A Virus M2 and M1 mRNAs

Abstract

Influenza A virus gene segment 7 encodes two proteins: the M1 protein translated from unspliced mRNA and the M2 protein produced by mRNA splicing and largely encoded by the M1 +1 reading frame. To better understand the generation of defective ribosomal products (DRiPs) relevant to MHC class I antigen presentation, we engineered influenza A virus gene segment 7 to encode the model H-2 Kb class I peptide ligand SIINFEKL at the M2 protein C-terminus. Remarkably, after treating virus-infected cells with the RNA splicing inhibitor spliceostatin A to prevent M2 mRNA generation, Kb-SIINFEKL complexes were still presented on the cell surface at levels of up to 60% of untreated cells. Three key findings indicate that SIINFEKL is produced by cytoplasmic translation of unspliced M1 mRNA initiating at CUG codons within the +1 reading frame: Synonymous mutation of CUG codons in the M2-reading frame reduced Kb-SIINFEKL generation.Kb-SIINFEKL generation was not affected by drug-mediated inhibition of AUG-initiated M1 synthesis.Kb-SIINFEKL was generated in vitro and in vivo from mRNA synthesized in the cytoplasm by vaccinia virus, and hence cannot be spliced. These findings define a viral DRiP generated by cytoplasmic non-canonical translation and demonstrate the participation of CUG-codon-based translation initiation in pathogen immunosurveillance.

Influenza A Virus Infection Induces Viral and Cellular Defective Ribosomal Products Encoded by Alternative Reading Frames

“…In summary, we show that what is very likely to be mistranslation of a nonfunctional ARF encoded by IAV leads to the rapid presentation of an epitope at or near the top of the antiviral T CD8+ immunodominance hierarchy. Along with the findings of Yang et al (58) demonstrating CUG initiation of a +1 RF model peptide after the M1 mRNA stop codon, this provides clear biological evidence for the importance of DRiPs in peptide generation, extending the voluminous evidence from studies of peptide generation in cultured cells.…”

Influenza A Virus Negative Strand RNA is Translated for CD8+ T Cell Immunosurveillance

“…Initiation could also occur on any of other 4 AUG codons, despite their low score (all at background levels), since the accuracy of the algorithm falls off at the bottom end, with a specificity of only 20%. In addition, we note the presence of 4 CUG codons that could support initiation, particularly since CUG initiation is likely favored during IAV infection (16, 33).…”

“…Though the biochemical nature of DRiPs generated from wild-type proteins remains largely undefined, viral peptides can clearly originate from non-canonical translation products (10). This includes downstream initiation on AUG codons (11), read through of stop codons (12, 13), frame shifting (14, 15), initiation on CUG or other alternative start codons (16), stop codon read through (12, 17) and translation of viral RNA in the nucleus (18). Inasmuch as each of these mechanisms would likely generate a defective gene product that is rapidly degraded, they provide a clear source of immunologically relevant DRiPs.…”

Accessory Gene Products of Influenza A Virus

“…As well as prompting the discovery of PB1-F2 ( promptly reclassified as a genuine accessory gene) (Chen et al 2001), specific DRiPs that provide T cell epitopes originating from both canonical (AUG) and noncanonical (CUG) internal translation initiation in the M1 and NS1 genes (Figs. 4B and 5A, respectively) have been identified (Yang et al 2016;Zanker et al 2019).…”