title: 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 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.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and.
authors.forename: Ning
authors.initials: N
authors.lastname: Yang
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and.
authors.forename: James S
authors.initials: JS
authors.lastname: Gibbs
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and.
authors.forename: Heather D
authors.initials: HD
authors.lastname: Hickman
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and.
authors.forename: Glennys V
authors.initials: GV
authors.lastname: Reynoso
authors.affiliation: Department of Chemistry, Purdue University, West Lafayette, IN 47907.
authors.forename: Arun K
authors.identifier: 0000-0003-2472-1841
authors.initials: AK
authors.lastname: Ghosh
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and.
authors.forename: Jack R
authors.initials: JR
authors.lastname: Bennink
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and jyewdell@niaid.nih.gov.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000187:drug effects; D002460:Cell Line; D003673:Defective Viruses / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005805:Genes, MHC Class I; D006367:HeLa Cells; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis; D011714:Pyrans / Q000494:pharmacology; D012326:RNA Splicing / Q000187:drug effects; D012270:Ribosomes / Q000378:metabolism*; D013141:Spiro Compounds / Q000494:pharmacology; D014763:Viral Matrix Proteins / Q000235:genetics*; D000086082:Viroporin Proteins
chemical_list: D010455:Peptides; D011714:Pyrans; D013141:Spiro Compounds; D014763:Viral Matrix Proteins; C488936:M1 protein, Influenza A virus; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C553684:spliceostatin A