Intracellular localization of proteasomal degradation of a viral antigen

To better understand proteasomal degradation of nuclear proteins and viral antigens we studied mutated forms of influenza virus nucleoprotein (NP) tha t misfold and are rapidly degraded by proteasomes. In the presence of prote asome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquiti nated and nonubiquitinated species in nuclear substructures known as promye locytic leukemia oncogenic domains (PODs) and the microtubule organizing ce nter (MTOC). Immunofluorescence revealed that dNP recruits proteasomes and a selective assortment of molecular chaperones to both locales, and that a similar (though less dramatic) effect is induced by proteasome inhibitors i n the absence of dNP expression. Biochemical evidence is consistent with th e idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhi bitors. Restoring proteasome activity while blocking protein synthesis resu lts in disappearance of dNP from PODs and the MTOC and the generation of a major histocompatibility complex class I-bound peptide derived from dNP but not NP. These findings demonstrate that PODs and the MTOC serve as sites o f proteasomal degradation of misfolded dNP and probably cellular proteins a s well, and imply that antigenic peptides are generated at one or both of t hese sites.