Ubiquitination is essential for human cytomegalovirus US11-mediated dislocation of MHC class I molecules from the endoplasmic reticulum to the cytosol.
Human cytomegalovirus encodes two glycoproteins, US2 and US11, which cause rapid degradation of MHC class I molecules, thus preventing recognition of virus-infected cells by the immune system. This degradation process involves retrograde transport or 'dislocation' of MHC class I molecules from the endoplasmic reticulum (ER) to the cytosol, where they are deglycosylated by an N-glycanase and degraded by the proteasome. At present it is unknown whether ubiquitination is required for US2- and US11-mediated dislocation and degradation of MHC class I molecules. Here, we show that in E36ts20 hamster cells, which contain a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme, US11-mediated degradation of MHC class I molecules is strongly impaired at the non-permissive temperature, indicating the necessity for ubiquitination in this process. We next addressed the question of whether ubiquitination is a condition for the retrograde movement of MHC class I molecules from the ER to the cytosol, or whether ubiquitination is merely required for recognition of dislocated MHC class I molecules by the proteasome. In the absence of a functional ubiquitin system, complexes of US11 and MHC class I molecules accumulate in the ER. In this state the membrane topology of MHC class I molecules does not significantly change, as judged from proteinase K digestions. Thus the results indicate that a functional ubiquitin system is essential for dislocation of MHC class I molecules from the ER to the cytosol.
Kikkert
M
M
Department of Medical Microbiology, Leiden University Medical Centre, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Hassink
G
G
Barel
M
M
Hirsch
C
C
van der Wal
F J
FJ
Wiertz
E
E
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003546:Cysteine Endopeptidases; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015789:HLA-A2 Antigen / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008025:Ligases / Q000235:genetics; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000502:physiology*; D013450:Sulfones / Q000494:pharmacology; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000502:physiology; D014764:Viral Proteins / Q000502:physiology*
D015789:HLA-A2 Antigen; D015395:Histocompatibility Antigens Class I; D009097:Multienzyme Complexes; D009842:Oligopeptides; D011480:Protease Inhibitors; D016601:RNA-Binding Proteins; D013450:Sulfones; C074546:US11 protein, herpesvirus; C118670:US2 protein, Varicellovirus; D014452:Ubiquitins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; C106727:carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes