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

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 relevant to MHC class I Ag presentation, we engineered influenza A virus gene segment 7 to encode the model H-2 K(b) 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, K(b)-SIINFEKL complexes were still presented on the cell surface at levels ≤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: 1) synonymous mutation of CUG codons in the M2-reading frame reduced K(b)-SIINFEKL generation; 2) K(b)-SIINFEKL generation was not affected by drug-mediated inhibition of AUG-initiated M1 synthesis; and 3) K(b)-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 defective ribosomal product generated by cytoplasmic noncanonical translation and demonstrate the participation of CUG-codon-based translation initiation in pathogen immunosurveillance.