Abstract
Polypeptides are organized into distinct substructures, termed protein domains, that are often associated with diverse functions. These modular units can act as binding sites, areas of post-translational modification, and sites of complex multimerization. The human cytomegalovirus US2 gene product is organized into discrete domains that together catalyze the proteasome-dependent degradation of class I major histocompatibility complex heavy chains. US2 co-opts the endogenous ER quality control pathway in order to dispose of class I. The US2 endoplasmic reticulum (ER)-lumenal region is the class I binding domain, whereas the carboxyl terminus can be referred to as the degradation domain. In the present study, we examined the role of the US2 transmembrane domain in virus-mediated class I degradation. Replacement of the US2 transmembrane domain with that of the CD4 glycoprotein completely blocked the ability of US2 to induce class I destruction. A more precise mutagenesis revealed that subregions of the US2 transmembrane domain differ in their ability to trigger class I degradation. Collectively, the data support a model in which US2-mediated class I degradation occurs as a highly regulated process where the US2 transmembrane domain and cytoplasmic tail work in concert to eliminate class I molecules. Host factors, including a signal peptidase complex, probably associate with the US2 molecule in a coordinated fashion to create a predislocation complex to promote the extraction of class I out of the ER. The results imply that the ER quality control machinery may recognize and eliminate misfolded proteins using a similar multistep regulated process.
Highlights
US2 and US11 have co-opted the cellular process known as endoplasmic reticulum (ER) quality control, which controls the proper assembly of nascent secretory proteins, in an effort to inhibit the cell surface presentation of class I MHC molecules [13, 14]
human cytomegalovirus (HCMV) US2 Transmembrane Domain Chimeras Are ER-localized Species—US2 is one of two HCMV-encoded ER-resident proteins, the other being US11, that exploit the process known as ER quality control to target class I MHC molecules for degradation [16]
To ascertain the role of the US2 transmembrane domain in class I degradation, US2 chimeric mutants were generated in which either the US2 transmembrane domain alone was exchanged for CD4 sequences (US2CD4US2) or both the transmembrane domain and cytoplasmic tail of US2 were replaced with CD4 sequences (US2CD4) (Fig. 1A)
Summary
Cells and Antibodies—Human U373-MG astrocytoma cells and U373 transfectants that stably express US2 mutant constructs (see below) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 8% fetal bovine serum, 1 mM HEPES, 100 units/ml penicillin, and 100 g/ml streptomycin at 37 °C (95% air, 5% CO2). Flow Cytometry Analysis—Quantitative flow cytometry analysis of surface-expressed class I MHC molecules was assessed using W6/32 or HLA-A2 (1.5 g of antibody/1 ϫ 106 cells), followed by incubation with Alexa 647-conjugated anti-mouse IgG (Molecular Probes). 12 h later, cells were washed twice in PBS at room temperature, fixed with methanol/acetone (1:1) for 10 min at 4 °C, washed twice with PBS, and blocked for 30 min at room temperature in PBG (phosphate-buffered saline solution containing 0.5% bovine serum albumin, 0.2% (v/v) fish gelatin) supplemented with 0.075% saponin and 4% normal goat serum. Cells were incubated for 1 h at room temperature with both anti-US2 and anti-BiP antibody (1:500 each in PBG plus 0.075% saponin). Images were generated using Adobe Photoshop 7.0 (Adobe Systems, Inc.)
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