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. Author manuscript; available in PMC: 2009 Feb 2.
Published in final edited form as: J Gen Virol. 2008 May;89(Pt 5):1122–1130. doi: 10.1099/vir.0.83516-0

ER chaperones participate in HCMV US2-mediated degradation of class I MHC molecules

Kristina Oresic 1, Domenico Tortorella 1
PMCID: PMC2634742  NIHMSID: NIHMS82883  PMID: 18420789

Summary

Human cytomegalovirus (HCMV) is a β-herpes virus that prevents the surface expression of class I MHC molecules in an attempt to escape recognition via cytotoxic CD8+ T cells. The HCMV US2 and US11 gene products induce class I down regulation during the early phase of HCMV infection by facilitating the destruction of class I heavy chains. The class I heavy chains are transported across the ER membrane and into the cytosol by a process referred to as dislocation followed by proteasome degradation. This process has striking similarities to the degradation of misfolded ER proteins mediated by ER quality control. Even though the major steps of the dislocation reaction have been characterized, the cellular proteins, specifically the ER chaperones involved in targeting class I for dislocation has not been fully delineated. To elucidate the chaperones involved in HCMV-mediated class I dislocation, we utilized a chimeric class I heavy chains with an affinity tag at its carboxy-terminus. Interestingly, US2 but not US11 continued to target the class I chimera for destruction suggesting a structural limitation for US11-mediated degradation. Association studies in US2 cells and in cells that express a US2 mutant, US2-186HA, revealed that class I specifically interacts with calnexin, BiP, and calreticulin. These findings demonstrate that US2-mediated class I destruction utilizes specific chaperones to facilitate class I dislocation. The data suggest a more general model in which the chaperones that mediate protein folding may also function during ER quality control to eliminate aberrant ER proteins.

Keywords: chaperones, ER degradation, HCMV US2 and US11, proteasome, ER quality control

Introduction

Many viruses utilize strategies to interfere with class I MHC antigen presentation in order to prevent the detection and clearance of infected cells (Hewitt, 2003, Tortorella et al., 2000). The class I MHC molecule is a stable trimeric complex consisting of a glycosylated heavy chain, β2-microglobulin, and an antigenic peptide that is recognized by a cytotoxic CD8+ T lymphocyte (CTL) (Townsend & Bodmer, 1989). HCMV is proposed to avoid CTL-induced killing by limiting the cell surface presentation of antigenic peptides. HCMV dedicates numerous gene products (US2, US3, US6, US11, US10, and UL82) that can either associate with class I and/or modulate class I antigen presentation (Lin et al., 2007, Trgovcich et al., 2006). Most of these gene products are expressed during the immediate early/early phase of HCMV infection indicating a critical time for HCMV to avoid immune detection.

The HCMV type I transmembrane glycoproteins US2 and US11 prevent surface expression of class I molecules by mediating the proteasome destruction of class I heavy chains (Wiertz et al., 1996a, Wiertz et al., 1996b). US2 and US11 induce the extraction of class I heavy chains from the ER into the cytosol by the AAA-ATPase p97-Npl4-Ufd1 complex (Ye et al., 2001). Upon exposure to the cytosol, class I heavy chains are deglycosylated by N-glycanase and then degraded by the proteasome. US2 and US11 mediate the degradation of class I heavy chains in a manner similar to how ER quality control disposes of misfolded ER proteins. In fact, human diseases that are caused by the degradation of defective proteins include lung diseases (i.e. cystic fibrosis), neurological diseases (i.e. Fabri disease), and diabetes mellitus (Aridor & Hannan, 2002). Essentially, ER quality control enables cells to cast away ER polypeptides that do not reach their proper native conformation due to inherent amino acid mutations or improper glycosylation (Hiller et al., 1996, Sitia & Braakman, 2003). The early events of viral mediated class I degradation and the proteins involved in this process have not yet been characterized. Therefore, US2- and US11-mediated class I degradation is a robust model system to study the host-pathogen interactions of HCMV proteins, but can also provide insight into the general mechanism of dislocation and degradation of aberrant ER proteins.

In this study, a class I heavy chain molecule fitted with an affinity tag at its carboxy-terminus (Puig et al., 2001) was stably expressed in US2- and US11-cells to identify ER chaperones that complex with class I heavy chains prior to their dislocation. We observed that US11 was incapable of mediating the degradation of the chimeric class I molecules; hence our studies focused on US2-mediated class I degradation. Association studies implicated the involvement of calnexin, calreticulin, and BiP in US2-mediated degradation of class I molecules. These results suggest for the first time that a specific chaperone complex participates in US2-mediated destruction of class I heavy chains. The data implies that the chaperones involved in protein folding/maturation may also act as ER quality control ‘sensors’ to target misfolded proteins for degradation.

Materials and Methods

Cell lines and antibodies

Human U373-MG astrocytoma cells and U373 transfectants that stably express class I heavy chains-CTAP molecules (see below) were maintained in DMEM (Oresic et al., 2006). Rabbit polyclonal anti-US2, anti-class I heavy chain, and anti-PDI were generated as described (Fiebiger et al., 2002, Rehm et al., 2001, Tortorella et al., 1998). The anti-gp96 serum was provided by Dr. Ploegh (MIT, Cambridge, MA). The monoclonal antibodies W6/32 (Parham et al., 1979) were purified from hybridoma cultured supernatant (Harlow et al., 1985). The monoclonal anti-calnexin antibody (AF8) (Hochstenbach et al., 1992) was a gift from Dr. Brenner (Harvard Medical School, Boston, MA). Anti-calreticulin was purchased from Assay Design and anti-BiP antibodies from BD Biosciences.

Generation of the HC-CTAP cDNA and retrovirus transduction

Class I heavy chain chimera containing a tandem affinity purification (TAP) tag (Gavin et al., 2002, Puig et al., 2001) at its carboxy-terminus (HC-CTAP) was generated. The TAP tag is comprised of the Staphylococcus aureus protein A IgG binding domain and calmodulin binding domain separated by the tobacco etch virus (TEV) protease cleavage site (Figure 1) (Rigaut et al., 1999). The class I heavy chain cDNA was sub-cloned into the CTAP vector, a gift from Dr. Katze (University of Washington, Seattle, WA) and shuttled into the retroviral vector, pLgPW (Oresic et al., 2006). HC-CTAP chimeric cDNA was transduced into U373 (U373HC-CTAP), US11 (US11HC-CTAP), US2 (US2HC-CTAP), and US2-186HA (US2-186HAHC-CTAP) cells (Oresic et al., 2006). EGFP-expressing cells were sorted by the Flow Cytometry Core Facility at Mount Sinai School of Medicine.

Figure 1. Expression of chimeric class I heavy chains (HC-CTAP) in U373 cells.

Figure 1

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A) Schematic representation of a tandem affinity tag (TAP) fused to the carboxy-terminus of class I heavy chains (HC-CTAP). TAP contains a Protein A binding domain and calmodulin binding domain separated by a TEV cleavage site. B) IgG-Sepharose (lanes 1-3 and 10-12), HC10 antibodies (lanes 4-6 and 13-15), and W6/32 antibodies (lanes 7-9 and 16-18) were incubated with NP-40 lysis buffer (denoted —), U373 cell lysates, and U373HC-CTAP cell lysates. The immunoprecipitates were resolved by SDS-PAGE and analyzed by an immunoblot using anti-heavy chain (lanes 1-9) and rabbit sera (lanes 10-18). *represents non-specific polypeptides.

Immunoprecipitation

Immunoprecipitation experiments were performed from one million cells unless otherwise noted and the data are representative of at least three independent experiments. The total cell lysates represent an equivalent of 350,000 cells. Cells were treated with proteasome inhibitor carboxybenzyl-leucyl-leucyl-leucyl vinyl sulfone (ZL3VS) (gift from Dr. Ploegh (MIT, Cambridge, MA)) as described. Cells were lysed in 1 ml of NP-40 lysis buffer (50mM Tris, pH 7.4, 150 mM NaCl, 20mM MgCl2, 0.5% (v/v) NP-40)/million cells containing protease inhibitors (1μM leupeptin, 1μg/ml aprotinin and 0.5μM PMSF). Cell lysates were incubated with IgG-Sepharose (GE Healthcare) or the respective antibody. The immunoprecipitates were washed with wash buffer (50mM Tris, pH 7.4, 150 mM NaCl, 5mM EDTA, 0.5% (v/v) NP-40). When TEV protease cleavage was performed, the precipitates were washed in wash buffer and then TEV cleavage buffer (10mM HEPES, 150mM NaCl, 0.25% NP-40, 0.5mM EDTA, 1mM DTT). IgG-Sepharose precipitates were resuspended in at least 3 times volume of TEV cleavage buffer and 10 units of TEV/106 cells overnight at 4°C. The polypeptides were resolved using SDS-PAGE and detected by a standard immunoblot protocol. The HC-CTAP polypeptides can be detected using rabbit serum due to the ability of the Protein A domain to bind immunoglobulins.

Pulse-chase analysis

U373HC-CTAP and US2HC-CTAP cells were subjected to pulse-chase analysis as previously described (Oresic et al., 2006). In brief, cells were labeled with [35S]methionine at 37°C for 15 min and chased up to 40 min. HC-CTAP molecules were recovered from NP-40 derived cell lysates with IgG-Sepharose and resolved using SDS-PAGE. The radioactive signal was detected from a dried gel exposed to autorad film for 48 hrs.

Results

Characterization of HC-CTAP in U373 cells

MHC class I is a 43 kDa type I ER membrane glycoprotein with a luminal domain and a cytosolic tail of 33 residues. In order to identify ER proteins involved in viral-mediated class I degradation, we fused a tandem affinity purification (TAP) tag (Gavin et al., 2002, Puig et al., 2001) to the carboxy-terminus of class I heavy chain (HC-CTAP) (Figure 1A). The HC-CTAP construct was transduced into U373 (U373HC-CTAP), US2 (US2HC-CTAP), and US11 (US11HC-CTAP) cells. The addition of the TAP tag (130 aa, ∼15kDa) to class I heavy chain (∼43kDa) would result in a polypeptide with a relative molecular mass of ∼58 kDa. The attachment of the affinity tag at the carboxy-terminus would most likely not interfere with protein complexes between class I and ER chaperones. Hence, this can allow for the identification of ER chaperones that play a role in class I destruction.

One important prerequisite for the utilization of HC-CTAP as a tool to isolate protein complexes would be that HC-CTAP contains a functional affinity tag and binds to immunoglobulin molecules. To that end, equal numbers of control (U373) cells and U373HC-CTAP cells were lysed in NP-40 lysis buffer and incubated with IgG-Sepharose (Figure 1). In order to determine whether HC-CTAP could mature into a properly folded class I molecule, lysates were incubated with an anti-class I antibody (W6/32) that recognizes properly folded class I molecules. In addition, HC-CTAP was recovered with an antibody (HC10) that reacts with unfolded class I heavy chains to verify the expression of the class I heavy chain portion of HC-CTAP. The W6/32 and HC10 immunoprecipitations were performed using Protein A agarose pre-bound to the respective antibody. This would prevent these anti-class I antibodies from binding to the Protein A binding domain of HC-CTAP. The precipitates were subjected to immunoblot analysis using anti-heavy chain serum (Figure 1B, lanes 1-9) and rabbit serum (Figure 1B, lanes 10-18). As expected, properly folded endogenous class I molecules were recovered with W6/32 from both cell lines (Figure 1B, lanes 8-9). Unfolded class I molecules were precipitated from U373HC-CTAP cells (Figure 1B, lanes 5 and 14). At steady state, the amount of unfolded class I in U373 cells was very minute (Figure 1B, lane 6). Properly folded molecules are minimally comprised of class I heavy chain and β2m. The excess class I heavy chains in U373HC-CTAP cells (Figure S1) depleted the β2m levels causing an increase in unfolded heavy chain levels.

Analysis of HC-CTAP molecules recovered using IgG-Sepharose, W6/32, and HC10 (Figure 1B, lanes 10-12, 13-15, and 16-18, respectively) demonstrated that the chimera interacts with an immunoglobulin molecule (Figure 1B, lane 11) and was recognized by anti-class I antibodies (Figure 1B, lanes 14 and 17). The specificity of IgG-Sepharose for Protein A binding domain of HC-CTAP (Figure S1) does not require the use of the calmodulin binding domain to recover strongly associated proteins to HC-CTAP. An immunoprecipitation using a purified mouse IgG immunoglobulin pre-bound to Protein A agarose was used to exclude the possibility that HC-CTAP molecules were recovered by binding non-specifically to the added immunoglobulins (data not shown). The data demonstrated that HC-CTAP has a functional Protein A affinity tag that does not prevent the chimera from folding into a mature class I molecule.

HC-CTAP is degraded in an US2-dependant manner

An essential characteristic for the utilization of HC-CTAP to define the mechanism of viral-mediated class I destruction is that HC-CTAP must be degraded in a US2- or US11-dependent manner. To that end, levels of HC-CTAP were analyzed in U373HC-CTAP, US2HC-CTAP, and US11HC-CTAP cells untreated or treated with the proteasome inhibitor, (ZL3VS) (2.5 μM, 12 hrs) (Figure 2). Total cell lysates were subjected to immunoblot analysis using rabbit serum (Figure 2A and 2B, lanes 1-4) and anti-gp96 serum (Figure 2A and 2B, lanes 5-8). The anti-gp96 immunoblot was used to verify equal loading of cell lysates. Similar amounts of HC-CTAP polypeptides were observed in untreated or inhibitor treated U373HC-CTAP cells (Figure 2A and 2B, lanes 1-2), a result consistent with protein stability. A significant increase of HC-CTAP was observed in US2HC-CTAP treated with proteasome inhibitor when compared to untreated cells (Figure 2A, lanes 3-4). The lack of a deglycosylated intermediate was due to treatment with low concentration of proteasome inhibitor. On the other hand, equivalent levels of HC-CTAP molecules were observed in US11HC-CTAP cells treated with or without proteasome inhibitor (Figure 2B, lanes 3-4) implying that HC-CTAP was not degraded in US11 cells. These results indicate that an extension of the class I cytoplasmic tail prevents US11-mediated degradation, while it has no impact on US2-mediated degradation. Therefore, US2 mediates the degradation of HC-CTAP molecules and these cells were used for subsequent experiments to study the role of chaperones in the degradation process.

Figure 2. US2 and not US11 targets HC-CTAP for proteasomal degradation.

Figure 2

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A)Total cell lysates from U373HC-CTAP and US2HC-CTAP cells untreated and treated with proteasome inhibitor ZL3VS (2.5μM, 12 hrs) were subjected to SDS-PAGE and submitted to a rabbit serum (lanes 1-4) and an anti-gp96 immunoblot (lanes 5-8). B) US11HC-CTAP cells were subjected to a similar experiment as in (A). The HC-CTAP, gp96 polypeptides, and the molecular weight standards are indicated.

To further characterize US2-mediated degradation of HC-CTAP, we examined whether a deglycosylated HC-CTAP is generated in US2HC-CTAP cells treated with proteasome inhibitor ZL3VS (20 μM) for up to 6 hours (Figure 3A). A diagnostic readout for dislocated heavy chains is the visualization of a deglycosylated faster migrating class I intermediate of ∼4 kDa upon inclusion of proteasome inhibitor (Wiertz et al., 1996b). The total cell lysates were submitted to immunoblot analysis using anti-heavy chain serum. Increased levels of glycosylated class I heavy chains as well as the deglycosylated HC-CTAP species were observed upon proteasome inhibition for at least 2 hrs (Figure 3A, lanes 3-6). The results demonstrate that the addition of a polypeptide on the carboxy-terminus of heavy chain does not interfere with US2-mediated dislocation of the class I substrate.

Figure 3. HC-CTAP molecules are degraded with fast kinetics in US2 cells.

Figure 3

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A) Cell lysates from US2HC-CTAP cells treated with proteasome inhibitor ZL3VS (20μM) for up to 6 hrs were submitted to immunoblot analysis using rabbit serum. B) U373HC-CTAP and US2HC-CTAP cells were labeled with 35S-methionine for 15 min and chased up to 40 min. The HC-CTAP molecules were recovered from cell lysates using IgG-Sepharose and resolved on an SDS-polyacrylamide gel. C) The recovered HC-CTAP molecules were quantified by densitometry and plotted as a percentage of HC-CTAP at the various chase times using 0 min chase point as 100%. The glycosylated HC-CTAP, deglycosylated HC-CTAP (HC-CTAP(−)CHO) polypeptides, and the molecular weight standards are indicated.

To determine the kinetics of HC-CTAP degradation in US2HC-CTAP cells, we performed a pulse-chase experiment. U373HC-CTAP and US2HC-CTAP cells were metabolically labeled with 35S-methionine for 15 min and chased up to 40 min (Figure 3B). HC-CTAP was recovered with IgG-Sepharose and subjected to SDS-PAGE. As expected, HC-CTAP was stable in U373HC-CTAP cells throughout the chase period (Figure 3B, lanes 1-4 and Figure 3C). In contrast, the amount of HC-CTAP molecules from US2HC-CTAP cells rapidly decreased during the chase period (Figure 3B, lanes 5-9 and Figure 3C). In US2 cells, the half-life of HC-CTAP (∼20 min) (Figure 3C) was slower than endogenous heavy chain that is about ∼3-5 min (Wiertz et al., 1996b). Consistent with published data, the chimeric class I molecules were degraded with slower kinetics than endogenous heavy chains (Fiebiger et al., 2002, Story et al., 1999, Tirosh et al., 2003). Nevertheless, these results imply that chimeric HC-CTAP molecules were targeted for the proteasomal degradation in US2-expressing cells. Therefore, HC-CTAP can be used to isolate protein complexes that potentially participate in the ER-to-cytosol extraction process.

HC-CTAP complexes with both wild-type US2 and the degradation mutant US2-186HA

To verify that HC-CTAP associated proteins from US2HC-CTAP cells are involved in the dislocation/degradation reactions, HC-CTAP was introduced into a cell line that expresses a US2 mutant (US2-186HA) that does not induce class I degradation (Figure 4) (Oresic et al., 2006). The cytoplasmic tail of US2 was replaced with the influenza hemagglutinin epitope tag generating the US2-186HA chimera. Total cell lysates from equal numbers of U373, U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP cells untreated or treated with the proteasome inhibitor ZL3VS (25 μM, 5 hrs) were analyzed by an immunoblot using HC10 antibodies (Figure 4A). As expected, only endogenously expressed class I heavy chains were observed from control (U373) cells that do not express HC-CTAP (Figure 4A, lanes 1-2). Similar amounts of HC-CTAP and class I heavy chains were observed in U373HC-CTAP and US2-186HAHC-CTAP cells independent of proteasome inhibition (Figure 4A, lanes 3-6). Only minute levels of HC-CTAP and endogenous class I heavy chains were observed in US2HC-CTAP cells and these class I levels increased upon treatment with proteasome inhibitor (Figure 4A, lanes 7-8). More importantly, the slightly faster migrating deglycosylated HC-CTAP species (HC(−)CHO) was observed in proteasome inhibitor treated US2HC-CTAP cells (Figure 4A, lane 8), demonstrating that the HC-CTAP was dislocated across the ER membrane. Overall, these data further show that a degradation defective US2 molecule, US2-186HA, is unable to induce destruction of either class I or HC-CTAP.

Figure 4. HC-CTAP chimera interacts with US2 proteins.

Figure 4

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A) Total cell lysates from U373, U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP untreated or treated with the proteasome inhibitor ZL3VS (20μM, 5 hrs) were subjected to immunoblot analysis using the monoclonal anti-heavy chain antibody HC10. B) HC-CTAP molecules recovered from U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP cells using IgG-Sepharose and total cell lysates were subjected to immunoblot analysis using rabbit serum (lanes 1-8) and anti-US2 serum (lanes 9-16). The glycosylated HC-CTAP, deglycosylated HC-CTAP (HC-CTAP(−)CHO), glycosylated endogenous heavy chain (HC), deglycosylated endogenous heavy chain HC(−)CHO, wild type US2, US2-186HA polypeptides, and the molecular weight standards are indicated.

In order to confirm that the US2 binding domain of class I was preserved in HC-CTAP molecules, we performed an association experiment from US2 and US2-186HA cells (Figure 4B). IgG-Sepharose was incubated with lysates from U373, U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP cells. Since HC-CTAP is degraded is US2 cells, an increased number of US2 cells (∼4X) was used to visualize the associated US2 polypeptides. The precipitates were analyzed by an immunoblot using rabbit serum (Figure 4B, lanes 1-8) and anti-US2 serum (Figure 4B, lanes 9-16). Total cell lysates were used as a control for the migration pattern of HC-CTAP (Figure 4B, lanes 5-8) and US2 polypeptides (Figure 4B, lanes 13-16). As expected, HC-CTAP molecules from U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP cells were successfully recovered with IgG-Sepharose (Figure 4B, lanes 2-4). Notably, both US2 and US2-186HA molecules co-precipitated with HC-CTAP (Figure 4B, lanes 11-12) demonstrating that both US2 and US2-186HA complexed with HC-CTAP. These results confirm that the luminal domain of HC-CTAP molecules is the US2 binding domain (Gewurz et al., 2001). More importantly, US2-186HAHC-CTAP cells serve as an ideal control cell line for protein specificity of HC-CTAP associated proteins in US2-mediated degradation.

ER chaperones complex specifically with class I molecules in an US2-dependent manner

A select set of chaperones including calnexin and calreticulin mediate the folding of class I heavy chains into a mature class I trimeric molecule (Harris et al., 1998, Pamer & Cresswell, 1998). To examine whether the chaperones that assist in class I protein folding may be also involved in their extraction from the ER, we performed association experiments in US2HC-CTAP cells (Figure 5). To allow for the stoichiometric analysis of the chaperones that interact with HC-CTAP in US2HC-CTAP cells, association experiments were performed in a manner to recovery equivalent numbers of HC-CTAP molecules from HC-CTAP expressing cells. HC-CTAP molecules recovered from U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP cells using IgG-Sepharose and U373HC-CTAP cell lysates were submitted to TEV protease digestion. The samples were subjected to immunoblot analysis using anti-calnexin (Figure 5, lanes 1-4) and anti-heavy chain antibodies (Figure 5, lanes 5-8). Interestingly, a significant amount of calnexin was recovered from US2HC-CTAP cells when compared to the calnexin precipitated from U373HC-CTAP or US2-186HAHC-CTAP cells (Figure 5, compare lane 2 vs. lanes 1 and 3). Equivalent levels of HC-CTAP lacking its protein A binding domain and containing its calmodulin binding domain (HCCBD) were observed and cannot account for the levels of calnexin recovered from the abovementioned cells (Figure 5, lanes 5-7). To confirm the calnexin/HC-CTAP interaction in US2HC-CTAP cells, an association experiment was performed using equal numbers of U373HC-CTAP and US2HC-CTAP cells (Figure S2). These data were consistent with Figure 4 and show that more calnexin co-precipitates with HC-CTAP from US2HC-CTAP cells than U373HC-CTAP cells. The minute amount of calnexin associated with HC-CTAP from U373HC-CTAP or US2-186HAHC-CTAP cells is not due to different levels of calnexin in these cells (Figure S3) or the egress of HC-CTAP from the ER. The HC-CTAP molecules were endoglycosidase H sensitive suggesting that they are ER resident species (data not shown). Altogether the data demonstrate that calnexin interacts specifically with the degradation substrate in an US2-dependent manner.

Figure 5. Calnexin interacts exclusively with HC-CTAP from US2HC-CTAP cells.

Figure 5

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HC-CTAP molecules recovered from U373HC-CTAP (1×106), US2HC-CTAP (4×106), and US2-186HAHC-CTAP (1×106) cells using IgG-Sepharose and U373HC-CTAP cell lysates were submitted to TEV protease cleavage. The released material and total cell lysates from U373HC-CTAP were subjected to immunoblot analysis using anti-calnexin (lanes 1-4) and anti-class I heavy chain antibodies (lanes 5-8). The calnexin, HC-CTAP lacking the protein A binding domain (HCCBD) polypeptides, and the molecular weight standards are indicated.

To investigate whether other ER chaperones such as calreticulin and BiP also complex with class I in an US2-dependent manner, HC-CTAP proteins were recovered from U373HC-CTAP, US2HC-CTAP, and US2-186HAHC-CTAP cells using IgG-Sepharose and subjected to TEV protease digestion (Figure 6). The samples were subjected to immunoblot analysis using anti-calreticulin (Figure 6, lanes 1-4), anti-BiP (Figure 6, lanes 5-8), and anti-heavy chain antibodies (Figure 6, lanes 9-12). Total cell lysates from U373HC-CTAP cells were used to verify the migration of the respective polypeptides (Figure 6, lanes 4, 8, and 12). The anti-heavy chain blot revealed that similar levels of HC-CTAP were recovered from the respective cells (Figure 6, lanes 9-11). Interestingly, a significant amount of calreticulin and BiP co-precipitated with HC-CTAP from US2HC-CTAP cells (Figure 6, lane 2 and 6) when compared to chaperone levels precipitated from U373HC-CTAP (Figure 6, lanes 1 and 3) or US2-186HAHC-CTAP cells (Figure 6, lanes 5 and 6). As with calnexin, similar levels of BiP were expressed in the HC-CTAP transductants (Figure S3). These data also confirm that expression of HC-CTAP does not induce an ER stress response as indicated by the comparable levels of BiP in all cell lines. Together, these results demonstrate that calnexin, calreticulin, and BiP form a tight complex with the HC-CTAP degradation substrate.

Figure 6. BiP and calreticulin interact with HC-CTAP from US2HC-CTAP cells.

Figure 6

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HC-CTAP molecules recovered from U373HC-CTAP (1×106), US2HC-CTAP (4×106), and US2-186HAHC-CTAP (1×106) cells using IgG-Sepharose were submitted TEV protease cleavage. The released material and total cell lysates from U373HC-CTAP were subjected to immunoblot analysis using anti-calreticulin (lanes 1-4), anti-BiP (lanes 5-8), and anti-class I heavy chain antibodies (lanes 9-12). The calreticulin, BiP, HC-CTAP, HC-CTAP lacking the protein A binding domain (HCCBD) polypeptides, and the molecular weight standards are indicated.

A similar experiment was performed in which the HC-CTAP precipitates were analyzed for PDI because is a highly expressed chaperone at millimolar levels (Noiva & Lennarz, 1992) and its involvement in dislocation (Tsai et al., 2001, Molinari et al., 2002). The anti-PDI immunoblot revealed that HC-CTAP molecules did not complex with PDI either in U373HC-CTAP or US2HC-CTAP cells. Collectively, the results implicate that specific chaperones are involved in the early phases of class I dislocation.

To further substantiate the interaction of calnexin and calreticulin with HC-CTAP in US2 cells, the association of HC-CTAP with these chaperones was examined under conditions of proteasome inhibition. HC-CTAP was recovered from U373HC-CTAP and US2HC-CTAP cells untreated or treated with proteasome inhibitor ZL3VS (2.5 μM, 12 hrs) with IgG-Sepharose (Figure 7). Upon cleavage with TEV protease, samples were resolved using SDS-PAGE and subjected to immunoblot analysis using anti-calnexin (Figure 7, lanes 1-4), anti-calreticulin (Figure 7, lanes 5-8), and anti-heavy chain antibodies (Figure 7, lanes 9-12). As expected, we observed an increase in HC-CTAP lacking a protein A binding domain (HCCBD) in US2HC-CTAP cells treated with proteasome inhibitor (Figure 7, compare lane 10 vs. lane 12). This increase in HC-CTAP translated into a significant recovery of both calnexin (Figure 7, lanes 2 and 4) and calreticulin (Figure 7, lanes 6 and 8). These results suggest that these chaperones continue to interact with ER resident HC-CTAP molecules until they are extracted out of the ER. Interestingly, in U373HC-CTAP cells, slightly more calnexin and calreticulin co-precipitated with HC-CTAP from cells treated with proteasome inhibitor (Figure 7, compare lanes 3 and 7 vs. lanes 1 and 5). Inhibition of the proteasome prevents the generation of peptides thereby blocking class I maturation and causing the accumulation of misfolded class I molecules in the ER. Collectively, the results furthermore verify that these specific chaperones participate in early events of proteasome-mediated degradation of class I heavy chains.

Figure 7. Proteasome-inhibition causes prolonged association of calnexin and calreticulin with HC-CTAP molecules from US2HC-CTAP cells.

Figure 7

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HC-CTAP molecules recovered from U373HC-CTAP (1×106) and US2HC-CTAP (4×106) cells untreated or treated with proteasome inhibitor ZL3VS (2.5μM, 12 hrs) using IgG-Sepharose were subjected to TEV protease cleavage. The released material was submitted to immunoblot analysis using anti-calnexin (lanes 1-4), anti-calreticulin (lanes 5-8), and anti-heavy chain antibodies (lanes 9-12). The calnexin, calreticulin, glycosylated HC-CTAP lacking the protein A binding domain (HCCBD), and the molecular weight standards are indicated.

Discussion

HCMV has evolved strategies to utilize existing cellular processes to escape immune detection (Loureiro & Ploegh, 2006). The current data extends the paradigm that HCMV co-opts host machinery to include the use of ER chaperone complexes to modulate class I antigen presentation. Following the recognition of class I molecules by HCMV US2, cellular chaperones are most likely utilized to assist in the dislocation of class I heavy chains out of the ER. Even though class I is degraded in a similar manner to a misfolded protein, the unusual feature of US2-mediated class I degradation is that the class I molecule is properly folded. Therefore, US2 must convince the ER quality control apparatus that class I is efficiently disposed of with the assistance of ER chaperones as degradation ‘sensory’ factors. Using an affinity tagged class I molecule, our data demonstrate that the ER chaperones calnexin, calreticulin, and BiP aid US2 in targeting class I for destruction. The strong association of the class I chimera with calnexin and BiP was confirmed using mass spectroscopy analysis. A large-scale recovery of HC-CTAP from US2HC-CTAP cells revealed that calnexin and BiP were some of the major polypeptides complexed with HC-CTAP (Figure S5). Calreticulin was not observed as a major polypeptide suggesting that it might be a minor component in the complex or is recruited only during a specific step of the pre-dislocation process. The results revealed that these chaperones strongly associate with HC-CTAP prior to its extraction from the ER. In fact, conditions of proteasome inhibition in control U373 cells that induced misfolded class I molecules also illustrated an increased association with calnexin and calreticulin (Figure 7). This supports a model that specific chaperones play a role in either the recognition or recruitment of class I for dislocation. It is possible that these chaperones can act on class I molecules after they are triggered for the destruction. However, since these chaperones also play a role in folding of class I molecules, we favor a model where they are involved in one of the early steps of triggering class I for destruction.

The interaction of HC-CTAP with BiP in US2 cells that express HC-CTAP polypeptidesc confirms the role of this chaperone in US2-mediated class I degradation (Hegde et al., 2006). BiP is an ER chaperone involved in processing of nascent polypeptides and prevents the mixing of ER lumenal contents with the cytosol. BiP can also reduce protein aggregation and is involved in the unfolded protein response (Friedlander et al., 2000, Hegde et al., 2006, Kabani et al., 2003, Nishikawa et al., 2001). During the dislocation process, the degradation substrate is probably partially unfolded prior to its transport through the dislocon. At this point, BiP would interact with the misfolded protein and assist in the dislocation of the substrate. Therefore, the interaction of BiP with the degradation substrate would contribute to the efficient removal of the misfolded proteins. Chaperones, general benefactors in protein folding, have a decisive role in directing misfolded proteins to the dislocon (Brodsky, 2007, McCracken & Brodsky, 2005). Eukaryotic cells have evolved to efficiently recognize structural features of polypeptides that have not achieved their native conformation and target these proteins for proteasomal destruction (Hochstrasser & Varshavsky, 1990, Johnston et al., 1998, Nishikawa et al., 2001, Whiteside et al., 1995). The ER chaperones BiP, calnexin, calreticulin, ERp57, and PDI participate in the recognition and dislocation of specific degradation substrates (Nishikawa et al., 2001, Plemper et al., 1997, Romisch & Schekman, 1992, Molinari et al., 2002). For example, calnexin is required for the degradation of pro-α factor in yeast (McCracken & Brodsky, 1996), while a mutant form of tyrosinase utilizes calreticulin and BiP for its extraction from the ER (Popescu et al., 2005). In addition, damaged glycoproteins are recognized by the ER-resident protein EDEM that interacts with misprocessed N-linked glycans and targets them for proteasomal degradation (Hosokawa et al., 2001, Molinari et al., 2003). Despite the implication of these proteins in the degradation process, selective chaperones may be assigned to the disposal of specific ER degradation substrates. Consistent with that idea, HCMV US2 appears to utilize a specific set of cellular chaperones to efficiently induce destruction of class I heavy chains.

The calnexin-calreticulin binding cycle participates in the proper folding of class I molecules (Pamer & Cresswell, 1998). It is not clear if these chaperones are recruited by US2 to class I or if they are initially part of the folding apparatus and remain bound to class I in an US2-dependent manner. Interestingly, calnexin and calreticulin have been proposed to act as “pre-protein stabilizers for the misfolded or aggregation-prone substrates that need to be discarded” (Brodsky, 2007). Therefore, they may have a propensity to recognize misfolded proteins. On the other hand, the specific chaperones involved in the extraction of misfolded proteins may be the same proteins involved in the folding of the specific polypeptides into their mature forms. In this case, the folding chaperones will have dual function as mediators of protein folding as well as “sensory” molecules that recognize misfolded proteins. Whether these misfolded proteins display structural elements that trigger their dislocation or the prolonged interaction between the chaperone and the ER protein signals its degradation is not yet clarified. Interestingly, the association of HC-CTAP with calnexin and calreticulin is most probably mediated by a structural property of class I proteins and rather than its lectin-binding specificity. An HC-CTAP molecule lacking its N-linked glycan continued to selectively interact with calnexin and calreticulin in an US2-dependent manner (data not shown). In addition, US2 molecules lacking an N-linked glycan continue to target class I for degradation (data not shown). Other factors such as binding affinity and possible number of N-linked glycans could also be important features in recognizing misfolded protein. To understand the molecular details of how these chaperones prepare a substrate for dislocation continues to remain an open question. Therefore, HCMV US2- and US11-mediated down regulation of class I heavy chains are ideal systems that afford us a unique opportunity to delineate the dislocation reaction as well as define the interaction between HCMV and its host.

Supplementary Material

1

Acknowledgement

We thank Dr. Hidde Ploegh for the critical reagents used in this study. These studies were supported by the National Institutes of Health Grant # AI060905.

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