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) that misfold and are rapidly degraded by proteasomes. In the presence of proteasome inhibitors, mutated NP (dNP) accumulates in highly insoluble ubiquitinated and nonubiquitinated species in nuclear substructures known as promyelocytic leukemia oncogenic domains (PODs) and the microtubule organizing center (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 in the absence of dNP expression. Biochemical evidence is consistent with the idea that dNP is delivered to PODs/MTOC in the absence of proteasome inhibitors. Restoring proteasome activity while blocking protein synthesis results 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 of proteasomal degradation of misfolded dNP and probably cellular proteins as well, and imply that antigenic peptides are generated at one or both of these sites.
Antón
L C
LC
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, USA.
Schubert
U
U
Bacík
I
I
Princiotta
M F
MF
Wearsch
P A
PA
Gibbs
J
J
Day
P M
PM
Realini
C
C
Rechsteiner
M C
MC
Bennink
J R
JR
Yewdell
J W
JW
D017951:Antigen Presentation / Q000187:drug effects; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002172:Canavanine / Q000494:pharmacology; D018385:Centrosome / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D018832:Molecular Chaperones / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D009975:Orthomyxoviridae; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D012995:Solubility; D014407:Tumor Cells, Cultured; D014452:Ubiquitins / Q000378:metabolism; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000956:Antigens, Viral; D004791:Enzyme Inhibitors; D015395:Histocompatibility Antigens Class I; D007976:Leupeptins; D018832:Molecular Chaperones; D009097:Multienzyme Complexes; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010446:Peptide Fragments; D014452:Ubiquitins; D014758:Viral Core Proteins; D002172:Canavanine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde