Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase

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Abstract
The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIINFEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for Kb-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-Kb fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of Kb-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t1/2 of the biosynthetic source of NA peptide is ∼5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.