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Originally published In Press as doi:10.1074/jbc.M601026200 on May 10, 2006

J. Biol. Chem., Vol. 281, Issue 28, 19395-19406, July 14, 2006
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A Structural Determinant of Human Cytomegalovirus US2 Dictates the Down-regulation of Class I Major Histocompatibility Molecules*Formula

Kristina Oresic{ddagger}, Vanessa Noriega§1, Laura Andrews§, and Domenico Tortorella§2

From the §Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029 and the {ddagger}Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, 1000 Ljubljana, Slovenia

Received for publication, February 2, 2006 , and in revised form, April 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human cytomegalovirus down-regulates cell surface class I major histocompatibility (MHC) molecules, thus allowing the virus to proliferate while avoiding detection by CD8+ T lymphocytes. The unique short gene product US2 is a 199-amino acid type I endoplasmic reticulum glycoprotein that modulates surface expression of class I MHC products by targeting class I heavy chains for dislocation from the endoplasmic reticulum to the cytosol, where they undergo proteasomal degradation. Although the mechanism by which this viral protein targets class I heavy chains for destruction remains unclear, the putative US2 cytoplasmic tail comprised of only 14 residues is known to play a functional role. To determine the specific residues critical for mediating class I degradation, a mutagenesis analysis of the cytoplasmic tail of US2 was performed. Using truncation mutants, the removal of only 4 residues (mutant US2195) from the US2 carboxyl terminus completely abolishes class I destruction. Furthermore, site-directed mutagenesis of the US2 cytoplasmic tail revealed that the most critical residues for class I-induced destruction, cysteine 187, serine 190, tryptophan 193, and phenylalanine 196, occurs every third residue. This experimental data supports a model that the US2 cytoplasmic tail is in a310 helical configuration. Such a secondary structure would predict that one side of the 310 helical cytoplasmic tail would interact with the extraction apparatus to facilitate the dislocation and subsequent destruction of class I heavy chains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian immune system has evolved several mechanisms to survey for the presence of intracellular invaders such as viruses. One of the primary means to detect a virally infected cell is through the class I major histocompatibility complex (MHC)3 (1). Class I MHC molecules are assembled within the endoplasmic reticulum (ER) and consist of a glycosylated heavy chain complexed with a light chain beta2-microglobulin and an antigenic peptide of 8-10 residues to form a stable trimeric molecule (2). The heterotrimer is competent to egress from the ER to the cell surface for recognition by CD8+ T lymphocytes. The class I MHC molecules alert the immune system to the presence of intracellular pathogens through the presentation of virally derived antigenic peptides. Recognition of the virus-infected cell by specific cytotoxic T lymphocytes induces the destruction of the infected cell allowing the host to clear the virus.

Given the important role that antigen presentation plays in the detection and clearance of infected cells, many viruses have devised strategies to interfere with the surface expression of class I MHC molecules (3, 4). One member of the beta-herpes virus family, the human cytomegalovirus (HCMV), employs numerous evasive strategies to hinder this arm of adaptive immunity and thereby avoid lysis of infected cells by cytotoxic T lymphocytes. HCMV expresses at least four gene products, all within the unique short (US) region of its genome (US3, US6, US2, and US11), that directly modulate class I MHC antigen presentation (5). The US3 glycoprotein transiently binds and retains peptide-loaded class I molecules within the ER, hindering their trafficking to the cell surface (6, 7). US6 complexes with the transporter associated with the antigen presentation complex, inhibiting binding of ATP and peptide translocation (8, 9). The single-pass type I transmembrane glycoproteins US2 (aa 199) and US11 (aa 215) target class I heavy chains for destruction by the proteasome (10, 11). These tactics attenuate the surface expression of pathogen-derived peptides to allow HCMV to remain hidden from the immune system for at least a limited period of time.

Expression of US2 or US11 viral gene products alone prevents the surface expression of class I molecules, a result that mimics an HCMV-infected cell (10, 11). US2 and US11 catalyze the extraction of class I heavy chains from the ER to the cytosol by a process referred to as dislocation or retrotranslocation. Upon exposure of class I to the cytosolic face of the ER membrane, the cytosolic enzyme N-glycanase removes its N-linked glycan (12, 13) and class I is then degraded by the proteasome. US2- and US11-mediated destruction of class I heavy chains occurs in a similar manner to how misfolded ER proteins are destroyed by the ER quality control machinery (14, 15). Proteins targeted for degradation in this manner include genetic mutants of {alpha}1-antitrypsin (16) and the cystic fibrosis transmembrane conductance regulator (17). Also included in this pool of ER molecules targeted for proteasomal degradation are proteins involved in the physiological regulation of sterol and fatty acid metabolism (18). Therefore, US2- and US11-mediated degradation of class I MHC can be used to delineate the mechanistic processes of ER quality control. In fact, proteasome degradation of an ER protein was initially characterized in US11- and US2-expressing cells (10, 11). The US2/US11 model cell system demonstrated that ER proteins slated for destruction are dislocated through the ER membrane, deglycosylated by N-glycanase, extracted from the ER membrane probably by the AAA-ATPase cdc48 complex (19), and degraded by the proteasome.

Despite the fact that US2 and US11 catalyze the degradation of class I heavy chains with similar kinetics, they carry out their analogous function quite differently. These two viral HCMV molecules have markedly different structural and co-factor requirements to successfully accomplish the down-regulation of surface class I MHC molecules. For example, 1) US2 may prefer to degrade properly folded class I molecules, whereas US11 is indifferent to the tertiary structure of class I molecules (20); 2) US2 and US11 induce the accumulation of markedly unique ubiquitinylated class I MHC intermediates. Properly folded class I molecules are modified with 3-5 ubiquitins in a US2-dependent manner, whereas US11 catalyzes the polyubiquitinylation of class I molecules (21, 22). 3) The ER resident protein derlin-1 is essential for US11-mediated class I degradation but not US2-induced class I destruction (23, 24), and 4) a crucial glutamine residue at position 192 in the US11 transmembrane domain is important for class I destruction, while the US2 cytoplasmic tail, aa 186-199, is responsible for the induction of class I degradation (23, 25, 26). These striking differences between US2- and US11-mediated class I destruction support a paradigm that the viral gene products utilize diverse sets of proteins to destroy class I heavy chains.

We sought to further identify the relevant regions of the US2 cytoplasmic tail that are important for class I destruction. A mutagenesis study generating truncation and site-directed mutants uncovered that the most vital residues within the US2 cytoplasmic tail occur every third residue within the 14-amino acid cytoplamsic tail. The spatial pattern of these critical residues suggests that they are positioned along one side of a helical conformation. The secondary structure consistent with the experimental data implies that the tail is in a 310 helical conformation. Therefore, our data supports a model in which the US2 cytoplamsic tail may conform to a 310-helix and interact with ER and/or cytosolic factors to induce the extraction and degradation of class I molecules.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 µg/ml streptomycin at 37 °C in a humidified atmosphere (95% air, 5% CO2). GP2-293 cells (BD Biosciences) were utilized to generate retroviruses (see below) and were cultured in media similar to U373 cells. Anti-glyceraldehyde-3-phosphate dehydrogenase was purchased from Upstate%20Biotechnology">Upstate Biotechnology. Rabbit polyclonal anti-US2 antibody and anti-class I heavy chain antibody was raised against the bacterially expressed lumenal domain of US2 (aa 15-140) and class I heavy chain (HLA-A2 allele, aa 25-366), respectively. Monoclonal antibodies W6/32 (27) were purified from hybridoma-cultured supernatant.

cDNA Constructs—The US2 site-directed cDNA constructs (US2D186A, US2C187A, US2N188A, US2L189A, US2S190A, US2M191A, US2M192A, US2W193A, US2M194A, US2R195A, US2F196A, US2F197A, US2V198A, US2C199A, US2F196D, US2F197D, and US2V198D) were generated from the ligation of two PCR fragments in which one fragment contained the mutated codon. The US2 carboxyl-terminal truncation cDNA constructs (US2198, US2197, US2196, US2195, US2194, US2192, US2190, US2188, and US2186) were amplified with a primer corresponding to the 5' end of the US2 cDNA and a 3' primer containing the respective US2 nucleotide sequence followed by the stop codon TAG. The chimeric US2186HA construct was generated from the ligation of a PCR product using a 5' primer corresponding to the US2 cDNA and a 3' primer containing the appropriate US2 sequence followed by the influenza hemagglutinin epitope tag sequence. All of the constructs were equipped with restriction sites that allowed for insertion into pcDNA3.1(+) and then were subsequently subcloned into the retrovirus vector pLgPW. pLgPW was derived from pLNCX (Clontech) in which the neomycin resistance selection marker was replaced by enhanced green fluorescent protein cDNA (P. Stern and H. Ploegh, Harvard Medical School).

Retrovirus Transduction—Retrovirus vectors containing the respective US2 cDNA constructs and the vesicular stomatitis virus-G envelope were transiently transfected simultaneously into GP2-293 cells (BD Biosciences) by a lipid-based protocol to produce a pseudo-typed Moloney murine leukemia virus amphotropic retrovirus. In brief, 2.5 µg of pLgPW:US2 constructs and 2.5 µg of a plasmid expressing vesicular stomatitis virus glycoprotein (BD Biosciences) were mixed with 8 µlof Lipofectamine 2000 (Invitrogen) in 500 µl of Opti-MEM (Invitrogen) and incubated at room temperature for 20 min. The DNA-Lipofectamine complex was added dropwise to GP2-293 cells (0.5 x 106) containing 1.5 ml of Dulbecco's modified Eagle's medium with 8% fetal calf serum and lacking antibiotics. The media was changed every 24 h for 2 days. Seventy-two hours post-transfection media containing retrovirus particles was filtered through a 0.45-µm filter and added to U373 cells with a final concentration of 8 µg/ml hexadimethrine bromide (Sigma). Enhanced green fluorescent protein expressing cells were sorted using a MoFlo high-speed cell sorter (Cytomation) (Mount Sinai Flow Cytometry Facility) 3-5 days post-infection.

Cell Lysis and Immunoprecipitation—U373 cells were lysed in Nonidet P-40 lysis mixture (50 mM Tris, pH 7.4, 150 mM NaCl, 20 mM MgCl2, 0.5% (v/v) Nonidet P-40, supplemented with 1.5 µg/ml aprotinin, 1 µM leupeptin, and 2 mM phenylmethylsulfonyl fluoride) followed by the removal of nuclei and insoluble material using centrifugation (10 min, 14,500 x g at 4 °C). The lysates were incubated with the respective antibody followed by the addition of Pansorbin cells (Calbiochem) for 1.5 h on a nutator at 4 °C. The Pansorbin cells were pelleted and washed 3 times with NET buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% (v/v) Nonidet P-40). The polypeptides were released from the Pansorbin cells with 1x SDS-sample buffer (50 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.02% bromphenol blue, 50 mM dithiothreitol), resolved using SDS-PAGE, and subjected to immunoblot analysis.

Metabolic Labeling of Cells and Pulse-Chase Analysis—Cells were detached by trypsin treatment, followed by starvation in methionine-/cysteine-free Dulbecco's modified Eagle's medium for 45 min at 37 °C. Cells were metabolically labeled with 500 mCi of [35S]methionine/cysteine (1,200 Ci/mM; PerkinElmer Life Sciences)/ml at 37 °C for the indicated times. For pulse-chase analysis, the cells were chased in Dulbecco's modified Eagle's medium containing an excess of 25 mM cold methionine for the indicated times. Cells were pelleted by centrifugation and lysed in 1% SDS (100 µl/1 x 106 cells) with at least three rounds of incubation at 95 °C for 5 min and vigorous perturbation followed by the addition of Nonidet P-40 lysis buffer to dilute SDS to <0.1%. The Nonidet P-40 lysis buffer was supplemented with 3 µl of normal mouse serum/ml, 3 µlof normal rabbit serum/ml, 1.5 mg/ml aprotinin, 1 µM leupeptin, and 2 mM phenylmethylsulfonyl fluoride. The cell lysates were initially subjected to a precleared step that involved the addition of 50 µl of Pansorbin cells/ml of lysate and incubation on a nutator for 1 h at 4°C. The Pansorbin cells were centrifuged at 14,500 x g for 2 min and the respective polypeptides were recovered by immunoprecipitation as described above. The radioactive signal within the polyacrylamide gel was intensified using Autofluor (National Diagnostics). The dried polyacrylamide gel was exposed to autoradiography film for 2 days at -80 °C.

Flow Cytometry Analysis—Quantitative flow cytometry analysis of surface expressed MHC class I molecules was assessed using W6/32 (1.5 µg of immunoglobulin/1 x 106 cells) followed by incubation with phycoerythrin-conjugated anti-mouse IgG (Molecular Probes) or Alexa 647-conjugated anti-mouse IgG (Molecular Probes). The fluorescence signal was measured from 10,000 cells using a Cytomics FC 500 Flow Cytometer (Beckman). The data were analyzed using Flow Jo software (Tree Star, Inc.). The levels of surface class I molecules are represented by plots of normalized cell number versus fluorescence signal. Normalized cell number was calculated by setting the maximum cell number at the respective peak fluorescence value to 100. The percentage of surface class I molecules was calculated using the mean surface class I signal from US2 cells versus control cells (U373 cells transduced with empty vector) multiplied by 100.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Specificity of the HCMV US2 Cytoplasmic Tail Is Essential for Class I Degradation—HCMV US2 is a type I membrane glycoprotein comprised of 199 amino acids (28) that mediates the proteasomal destruction of class I MHC heavy chains (11, 29). US2 consists of an ER lumenal class I domain (aa 1-160), a transmembrane domain (aa 161-185), and a putative cytoplasmic tail of 14 residues (aa 186-199) (Fig. 1A) (30, 31). A US2 molecule that lacks its cytoplasmic tail (US2186) does not induce class I destruction but continues to tightly complex with class I molecules (25, 26). Hence, the US2 cytoplasmic tail is an important domain required for class I heavy chain destruction.

To address if the specific peptide sequence of the US2 cytoplasmic tail is important for class I destruction, we generated a U373 cell line ("Experimental Procedures") that expresses a chimeric US2 molecule in which the cytoplasmic tail of US2 was replaced with the influenza hemagglutinin epitope tag (US2186HA) (Fig. 1A). We examined the fate of newly synthesized class I heavy chains in US2186HA cells by pulse-chase analysis (Fig. 1B). The class I heavy chains (Fig. 1B, lanes 1-6) and US2 molecules (Fig. 1B, lanes 7-12) were recovered using their respective antisera. Similar levels of class I heavy chains were recovered throughout the chase periods from the control U373 and US2186HA cells (Fig. 1B, lanes 1-2 and 5-6) implying that US2186HA does not target class I for destruction. Results indicative of class I destruction were demonstrated by a decrease in class I heavy chains recovered from US2199 cells at the 0-min chase period and the lack of class I recovered from the 30-min chase period (Fig. 1B, lanes 3 and 4). Due to the kinetics of class I degradation and the pulse time (15 min), the class I synthesized during the pulse period is partially degraded prior to the start of the chase, hence less heavy chains are recovered at the 0-min chase period in US2199 cells compared with control cells. US2 wild type amended with the HA tag (US2199HA) continues to down-regulate class I molecules excluding the possibility that the HA tag can inhibit class I degradation.4 To verify that the lack of class I destruction in US2186HA cells was not due to expression levels of the US2 polypeptides, equivalent levels of US2199 and US2186HA were recovered from the respective cell lysates (Fig. 1B, lanes 9-12). Our results demonstrate that US2186HA does not induce the destruction of newly synthesized class I molecules.

Does US2186HA down-regulate surface class I MHC molecules? To address this question, we examined the cell surface class I molecules in US2186HA cells by flow cytometry (Fig. 1C) ("Experimental Procedures"). Control (U373) and wild type US2199 cells were used as negative and positive controls for class I MHC down-regulation. An immunoglobulin isotype control (solid line) was utilized to examine the background fluorescence signal (Fig. 1C, upper panel). A similar isotype control signal was observed in all subsequent flow cytometry experiments. As expected, a decrease of surface class I molecules was observed from US2199 cells (dashed line) when compared with control U373 cells (shaded) (Fig. 1C, upper panel). On the other hand, similar levels of surface class I molecules were exhibited on both US2186HA (solid line) and U373 cells (shaded) (Fig. 1C, lower panel) providing further evidence that US2186HA does not induce class I destruction. These data indicate that flow cytometry is a reliable assay to measure US2-mediated class I degradation.


Figure 1
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  		FIGURE 1.
US2186HA chimera does not induce class I destruction. A, wild type US2199 is a type I membrane protein consisting of a 160-amino acid luminal domain, a transmembrane domain between residues 160 and 185, and a short cytoplasmic tail of 14 residues. The US2 chimeric mutant US2186HA consists of the influenza HA epitope tag in place of the US2 cytoplasmic tail. The single letter amino acid code is used to identify the respective residue. B, U373, wild type US2199, and US2186HA cells were metabolically labeled with [35S]methionine for 15 min and chased up to 30 min. The class I heavy chains and US2 polypeptides (US2199 and US2186HA) were recovered from SDS-denatured cell lysates using anti-heavy chain (lanes 1-6) and anti-US2 sera (lanes 7-12). The immunoprecipitates were resolved on an SDS-polyacrylamide gel (12.5%). The glycosylated class I heavy chains (Class I HC), US2 polypeptides US2199 and US2186HA, and the molecular weight standards are indicated. C, flow cytometry analysis reveals that US2186HA does not down-regulate surface class I molecules. U373 (control) cells (shaded), US2199 (dashed line), and US2186HA (solid line) were labeled with W6/32 followed by an anti-mouse Ig conjugated to Alexa 647. U373 cells were labeled by an immunoglobulin isotype control (upper panel, solid line). The Alexa 647 fluorescence of surface class I is represented as normalized cell number versus fluorescence signal ("Experimental Procedures"). D, properly folded class I molecules were recovered by W6/32 (lanes 1-3) and US2 proteins were immunoprecipitated (IP) with anti-US2 serum (lanes 4-6) from U373, wild type US2199, and US2186HA cells. The polypeptides were resolved on an SDS-polyacrylamide gel (12.5%) and subjected to an immunoblot (IB) analysis using anti-heavy chain and anti-US2 sera. The migration position of class I heavy chains (class I HC), US2199, and US2186HA polypeptides are indicated. The immunoglobulin heavy chain (Ig HC), light chain (Ig LC), and molecular standards are noted.

 
To exclude the possibility that the inability of US2186HA to induce class I destruction is due to folding defects within US2186HA, we performed an US2/class I association experiment. Properly folded class I molecules and US2 polypeptides were recovered from U373, US2199, and US2186HA cells using W6/32 (Fig. 1D, lanes 1-3) and rabbit anti-US2 polyclonal serum (Fig. 1D, lanes 4-6), respectively. The precipitates were subjected to an immunoblot using rabbit anti-class I heavy chain and anti-US2 sera. We obtained equivalent amounts of class I heavy chains from U373 and US2186HA cells (Fig. 1D, lanes 1 and 3), whereas less class I was recovered from US2199 cells (Fig. 1D, lane 2). US2186HA was observed to co-precipitate with class I MHC molecules indicating that US2186HA associates with class I (Fig. 1D, lane 3). We did not observe US2199 co-precipitation with class I molecules (Fig. 1D, lane 2). This is most likely due to the relatively small amount of class I recovered in the W6/32 precipitates. US2199 and US2186HA polypeptides were precipitated from the respective cell lines (Fig. 1D, lanes 4-6). Collectively, the data demonstrate that US2186HA does not target class I for degradation because it lacks the necessary residues within its cytoplasmic tail.


Figure 2
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  		FIGURE 2.
Expression of US2 carboxyl-terminal truncation mutants in U373 cells. A, wild type US2199 and US2 carboxyl-terminal truncation mutants US2198, US2197, US2196, US2195, US2194, US2192, US2190, US2188, and US2186 lacking carboxyl-terminal acids are depicted in the schematic diagram. The single letter amino acid code is used to identify the respective residue. B, SDS cell lysates of U373 (control), US2186, US2188, US2190, US2192, US2194, US2196, US2198, and wild type US2199 cells were resolved on an SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-class I heavy chain serum (lanes 1-9), anti-US2 serum (lanes 10-18), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(lanes 19-27). The respective polypeptides and molecular mass standards are indicated. The asterisk (*) represents a nonspecific polypeptide.

 
Identification of the HCMV US2 Cytoplasmic Tail Region Important for Class I Degradation—Specific regions of the putative US2 cytoplasmic tail are likely responsible for mediating class I destruction (Fig. 1). To identify the cytoplasmic tail region necessary to induce class I destruction, we constructed US2 carboxyl-terminal truncation mutants US2186, US2188, US2190, US2192, US2194, US2196, and US2198 (Fig. 2A). The levels of class I MHC and US2 were examined from equivalent numbers of U373, wild type US2199, US2198, US2196, US2194, US2192, US2190, US2188, and US2186 cells using an anti-class I heavy chain (Fig. 2B, lanes 1-9), and an anti-US2 immunoblot (Fig. 2B, lanes 10-18). An anti-glyceraldehyde-3-phosphate dehydrogenase immunoblot was used as a loading control (Fig. 2B, lanes 19-27). The N-linked glycan acquired by class I MHC molecules is subjected to post-translation modifications in the secretory compartments yielding class I molecules that migrate as two populations on a highly cross-linked SDS-polyacrylamide gel (15%) (Fig. 2B, lanes 1-9). The data reveals that US2198 targets class I for destruction with similar effectiveness as US2199. Interestingly, US2196 also targets class I for destruction, albeit less efficiently than wild type US2. In contrast, US2188, US2190, US2192, and US2194 cells have equivalent levels of class I as control (U373) cells. The data suggests that the removal of only three residues from the carboxyl terminus can alter the ability of US2 to induce class I destruction.

The expression of US2 polypeptides in the respective cell lines was validated by an anti-US2 immunoblot (Fig. 2B, lanes 10-18). As expected, US2 polypeptides were not observed in U373 cells (Fig. 2B, lane 10). The US2 carboxyl-terminal truncation mutants migrated slightly faster in a stepwise fashion when compared with wild type US2199 (Fig. 2B, lanes 2-9) confirming their expression. We observed almost equivalent levels of US2 polypeptides with the exception of US2198. These results suggest that only a small amount of US2 polypeptide is required to target class I for destruction. Overexpression of a mutant such as US2194 cannot compensate for its inability to induce class I destruction.


Figure 3
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  		FIGURE 3.
Analysis of class I surface expression in cells expressing US2 carboxyl-terminal truncation mutants. A, the surface expression of class I MHC molecules on U373 (control), US2186, US2188, US2190, US2192, US2194, US2196, US2198, and wild type US2199 cells was analyzed by flow cytometry. U373 (control) cells (solid line) and US2-expressing cells (dashed line) were labeled with W6/32 followed by an anti-mouse Ig conjugated to phycoerythrin. The signal of surface class I molecules is represented as normalized cell number versus fluorescence signal ("Experimental Procedures"). B, properly folded class I molecules were recovered by W6/32 (lanes 1-9), U373 (control), US2199, US2198, US2196, US2194, US2192, US2190, US2188, and US2186 cells. The precipitates were resolved on an SDS-polyacrylamide gel (12.5%) and subjected to immunoblot analysis using anti-heavy chain and anti-US2 sera. Because class I is degraded in US2199, US2198, and US2196 cells, class I molecules were recovered from 4 times more US2199 and US2198 cells and 2.5 times more US2196 cells than the US2 mutant cells to visualize equivalent levels of class I heavy chains in all samples. The migration position of class I heavy chains (class I HC), US2 proteins, and molecular mass standards are indicated.

 
To verify the ability of the US2 truncation mutants to down-regulate class I molecules, we subjected U373, US2186, US2188, US2190, US2192, US2194, US2196, US2198, and wild type US2199 cells to flow cytometry (Fig. 3A). A significant reduction of surface class I was observed in cells that express US2199 and US2198 (~90%), whereas the surface class I in US2196 cells was modestly reduced (~70%). The intermediate result observed in US2196 cells suggests that class I is probably degraded at a slower rate. In contrast, the levels of class I molecules observed from US2194, US2192, US2190, US2188, and US2186 cells were similar to that of U373 cells (Fig. 3A). These results demonstrate that the four amino acids (aa 196-199) carboxyl-terminal are involved in class I degradation.

To confirm that the class I binding domain of the US2 carboxyl-terminal truncation mutants was not altered, we performed an US2/class I association experiment. Class I MHC molecules were recovered from U373, wild type US2199, US2198, US2196, US2194, US2192, US2190, US2188, and US2186 cells using W6/32. The immunoprecipitates were subjected to SDS-PAGE and then analyzed by an immunoblot using anti-US2 and anti-class I heavy chain sera (Fig. 3B). As expected, class I heavy chains were recovered from the W6/32 immunoprecipitates (Fig. 3B, lanes 1-9). Because class I is degraded in US2199, US2198, and US2196 cells, class I molecules were recovered from an increased number of cells to visualize equivalent amounts of class I heavy chains in all samples. All of the respective US2 polypeptides co-precipitated with class I molecules (Fig. 3B, lanes 2-9). Thus, we conclude that the carboxyl-terminal deletion mutants that did not induce class I degradation was not due to a defective class I binding domain.

To further delineate the US2 cytoplasmic tail length requirement for class I MHC destruction, we generated U373 cells that stably express the carboxyl-terminal truncation mutants US2195 and US2197 (Fig. 4A). An immunoblot using anti-US2 serum confirmed that these molecules were expressed in the respective cell line (supplemental Fig. 1). As with the other carboxyl-terminal truncation mutants, US2197 and US2195 continued to associate with class I molecules.4 U373, US2199, US2197, and US2195 cells were subjected to flow cytometric analysis using W6/32 and anti-mouse Ig Alexa 647 (Fig. 4B). A reduction of surface class I was observed in cells that express US2199 and US2197 implying that US2197 down-regulates class I to an equivalent level as US2199. On the other hand, similar levels of class I were observed on the surface of US2195 and U373 cells indicating that US2195 lacks critical residues to induce class I degradation.


Figure 4
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  		FIGURE 4.
Removal of 4 residues from the cytoplamsic tail of US2 (US2195) inactivates class I degradation function. A, wild type US2199 and US2 carboxyl-terminal truncation mutants US2197 and US2195 lacking carboxyl-terminal acids are depicted in the schematic diagram. The single letter amino acid code is used to identify the respective residue. B, the surface expression of class I MHC molecules on U373 (control), US2199, US2197, and US2195 cells was analyzed by flow cytometry. U373 (control) cells (solid line) and US2-expressing cells (dashed line) were labeled with W6/32 followed by an anti-mouse Ig conjugated to Alexa 647. The Alexa 647 fluorescence of surface class I is represented as normalized cell number versus fluorescence signal ("Experimental Procedures").

 
In summary, the US2 truncation data implicates phenylalanine 196 and 197 as potentially key amino acids for the efficient induction of class I destruction (Table 1). The removal of the carboxyl-terminal residues, cysteine 199 and valine 198, does not alter the ability of US2 to degrade class I suggesting that these residues are not necessary for class I destruction. However, removal of only three residues (US2196) has a significant effect on class I destruction and lack of four residues (US2195) completely abolishes its ability to induce class I destruction. It is not clear if phenylalanines 196 and 197 are directly involved in class I degradation or if they provide structural support for different regions of the cytoplasmic tail.


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  		TABLE 1
Class I MHC degradation by US2 truncation mutants

 
Analysis of Site-directed Alanine Mutants of the US2 Cytoplasmic Tail—To address if specific residues within the US2 cytoplasmic tail are involved in US2-induced class I down-regulation, we generated site-directed US2 mutants in which every residue of the US2 cytoplasmic tail (aa 186-199) was replaced with alanine: US2D186A, US2C187A, US2N188A, US2L189A, US2S190A, US2M191A, US2M192A, US2W193A, US2M194A, US2R195A, US2F196A, US2F197A, US2V198A, and US2C199A (Fig. 5A). U373 cells that stably express the respective site-directed US2 constructs were verified by an anti-US2 immunoblot.4 We utilized flow cytometry to examine the ability of the US2 site-directed mutants to down-regulate class I (Fig. 5B). US2C187A, US2S190A, and US2W193A cells expressed equivalent levels of class I as U373 cells. On the other hand, US2D186A, US2N188A, US2L189A, US2M191A, US2M192A, US2M194A, US2R195A, US2F196A, US2F197A, US2V198A, and US2C199A down-regulated surface class I molecules to similar levels of US2199 (Fig. 5B). Quantitation of five independent experiments revealed that US2C187A, US2S190A, and US2W193A down-regulated only about 20% of class I molecules implying that these residues are directly involved in targeting class I for destruction (Fig. 5C). In contrast, US2D186A, US2N188A, US2L189A, US2M191A, US2M192A, US2M194A, US2R195A, US2V198A, and US2C199A down-regulated at least 80% of the surface class I molecules. Interestingly, US2F196A and US2F197A displayed an intermediate phenotype such that they down-regulated only ~64 and ~77% of class I molecules, respectively. These results suggest that Phe196 and Phe197 may also play a role in class I degradation. To confirm the modest down-regulation of US2F196A and US2F197A, we examined the steady state levels of class I in U373, US2199, US2F196A, US2F197A, US2V198A, and US2C199A cells by an antiheavy chain immunoblot (supplemental Fig. 2). We observed increased amounts of class I from US2F196A and US2F197A cells when compared with US2199, US2V198A, and US2C199A cells. These results are consistent with the flow cytometry data and further suggest a role for phenylalanines 196 and 197 in class I down-regulation. Collectively, the results suggest a hierarchy of critical residues in which cysteine 187, serine 190, and tryptophan 193 can be the most essential residues involved in class I down-regulation.


Figure 5
Figure 5
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  		FIGURE 5.
Analysis of class I surface expression in cells expressing US2 site-directed alanine mutants. A, wild type US2199 and US2 site-directed mutants US2C199A, US2V198A, US2F197A, US2F196A, US2R195A, US2M194A, US2W193A, US2M192A, US2M191A, US2S190A, US2L189A, US2N188A, US2C187A, and US2D186A are depicted in a schematic diagram. The single letter amino acid code is used to identify the respective residue. B, the surface expression of class I MHC molecules on U373 (control), US2199, US2C199A, US2V198A, US2F197A, US2F196A, US2R195A, US2M194A, US2W193A, US2M192A, US2M191A, US2S190A, US2L189A, US2N188A, US2C187A, and US2D186A cells was analyzed by flow cytometry. U373 (control) cells (shaded), US2199 cells (solid line), and US2 mutant-expressing cells or Ig control (dashed line) were labeled with W6/32 followed by an anti-mouse Ig conjugated to Alexa 647. The Alexa 647 fluorescence of surface class I molecules is represented as normalized cell number versus fluorescence signal ("Experimental Procedures"). C, the amount of surface class I molecules (%) of the site-directed mutants was calculated from five independent experiments using the mean value of surface class I of US2 expressing cells versus U373 cells x100. The standard deviation of the % surface class I is represented by error bars.

 
Analysis of Site-directed Aspartic Acid Mutants of the US2 Cytoplasmic Tail—The truncation mutagenesis study proposes that phenylalanines 196 and 197 may be involved in class I degradation (Figs. 3 and 4) even though the site-directed alanine mutant data does not necessarily implicate these residues in participating in class I down-regulation. The modest down-regulation of class I by US2F196A and US2F197A (Fig. 5) was not surprising because the two phenyl-alanines were replaced with a conservative alanine residue. Therefore, to address whether these residues play a direct role in class I down-regulation, we generated site-directed mutants in which hydrophobic residues phenylalanine 196 and 197 were replaced with aspartic acid (US2F196D and US2F197D) (Fig. 6A). We also generated an additional site-directed aspartic acid mutant US2V198D (Fig. 6A) as a control to exclude the possibility that the hydrophobic patch created by residues 196-198 (FFV) would be essential for class I down-regulation. The stable expression of US2F198D, US2F197D, and US2V198D was confirmed by an anti-US2 immunoblot (supplemental Fig. 1). The surface expression of class I MHC was examined in U373, US2199, US2F198D, US2F197D, and US2V198D cells by flow cytometry using W6/32 and anti-mouse Ig Alexa 647 (Fig. 6B). US2F197D and US2V198D cells down-regulated surface class I molecules at similar levels as wild type US2199. This suggests that phenylalanine 197 and valine 198 are not essential for class I degradation. In contrast, similar levels of surface class I molecules from U373 (control) and US2F196D cells were observed (Fig. 6B). These results demonstrate that phenylalanine 196 of the US2 tail is important for class I down-regulation. Collectively (Table 2), the mutagenesis results reveal that every third residue from cysteine 187 to phenylalanine 196 is involved in the degradation of class I molecules.


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  		TABLE 2
Class I MHC degradation by US2 site-directed mutants

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The human cytomegalovirus US2 glycoprotein binds to class I molecules and subsequently triggers its dislocation and proteasome-mediated degradation. The interaction of the US2 amino-terminal ER lumenal domain (aa 1-160) with class I alone does not induce the destruction of class I heavy chains and hence an alternate US2 region is required. The US2 domain that has been implicated as the degradation domain is the carboxyl-terminal end of the molecule, specifically its 14-residue cytoplasmic tail (aa 186-199). A US2 lacking its cytoplasmic tail continues to complex with class I, but does not bring about the degradation of class I molecules (Fig. 1 and Refs. 25 and 26). We propose that US2-mediated class I destruction occurs in a series of steps initiated by the interaction of US2 with class I MHC molecules (Fig. 7A). The tight interaction of class I with US2 allows the cytoplasmic tail to engage the dislocation machinery and induce class I dislocation. Our data demonstrates that the US2 cytoplasmic tail residues cysteine 187, serine 190, tryptophan 193, and phenylalanine 196 most likely interact with the dislocation apparatus and trigger the machinery to extract class I from the ER.

How does the ER lumenal event of US2/class I interaction activate the cytoplasmic tail to trigger class I dislocation? HCMV US2 appears to have taken a lesson from the signal transduction process and the ER stress response in which a protein-protein interaction on one side of a membrane can initiate a cascade of events on the opposite side of the bilayer. Receptors on the cell surface associating with specific ligands such as growth factors or cytokines can activate transcription factors and other cytoplasmic events such as signal transduction pathways (32). In a similar manner, ER stress caused by an accumulation of misfolded ER proteins can induce up-regulation of ER chaperones via activation of the transcription factor XBP-1, or by directly triggering translation attenuation through the phosphorylation of eukaryotic initiation factor 2{alpha} (33). In the case of US2, the molecular basis for dislocation of class I is likely due to the recruitment of soluble cytosolic factors and/or ER membrane proteins by its cytoplasmic tail to initiate the dislocation process. One possible model is that the association of US2 with class I promotes a conformational change within the US2 tail allowing it to engage the dislocation apparatus. An alternative possibility is that the US2 tail is in a relaxed state and upon the interaction of US2 with class I; the US2 cytoplasmic tail is stabilized into a conformation capable of associating with the dislocation complex. What continues to remain a mystery is how US2 notifies the quality control machinery to extract class I heavy chains from the ER.


Figure 6
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  		FIGURE 6.
Analysis of class I surface expression in cells expressing US2 site-directed aspartic acid mutants. A, wild type US2199 and US2 site-directed mutants US2V198D, US2F197D, and US2F196D are depicted in a schematic diagram. The single letter amino acid code is used to identify the respective residue. B, the surface expression of class I MHC molecules on U373 (control), US2199, US2V198D, US2F197D, and US2F196D cells was analyzed by flow cytometry. U373 (control) cells (solid line) and US2-expressing cells (dashed line) were labeled with W6/32 followed by an anti-mouse Ig conjugated to Alexa 647. The Alexa 647 fluorescence value of surface class I molecules is represented as the normalized cell number versus fluorescence signal ("Experimental Procedures").

 
The US2 mutagenesis study demonstrates that the US2 cytoplasmic tail residues cysteine 187, serine 190, tryptophan 193, and phenylalanine 196 are important for class I down-regulation (Figs. 5 and 6). The site-directed mutants US2C187A, US2S190A, US2W193A, and US2F196D down-regulate less than ~20% of class I molecules (Figs. 5 and 6) when compared with wild type US2 (~90%). The loss of only one residue had a significant detrimental effect on class I degradation. Consistent with the site-directed mutagenesis data is the truncation mutant results (Figs. 3 and 4) that demonstrate that phenylalanines 196 and 197 are involved in class I degradation. Under these circumstances, the cytoplasmic tail of US2196 was probably in an unstable conformation and only required an additional residue in the form of phenylalanine 197 to stabilize and trigger class I destruction with similar effectiveness as wild type US2.

Given the likelihood that the US2 tail associates with a molecular cleft in a polypeptide of the dislocon complex, we predict that these residues are along the same surface of the US2 tail structure to enable an interaction in a highly specific manner with the dislocation machinery. A secondary structure that corresponds to such a configuration is the 310-helix. The 310-helix has three residues per turn as compared with the classical{alpha}-helix in which 3.6 residues revolve around the helical axis (34). A 310-helix can occur at the termini of an {alpha}-helix, a property consistent with the US2 cytoplamsic tail because it follows the transmembrane domain that most likely is an {alpha}-helical structure. Most 310 helices within the globular structure of a protein have a mean length of 3.3 residues (34); clearly this property of a classical 310-helix differs from the 14-residue US2 cytoplasmic tail. Most of the characterized 310 helices are found within crystallized globular proteins. Therefore, the existence of a longer 310-helix at the carboxyl-terminal of a polypeptide may be technically challenging to define because of its instability at the termini of polypeptides. However, we provide experimental data suggesting that the active form of the US2 cytoplamsic tail may adopt a 310 helical structure (Fig. 7B).

The 310-helix comprises a small, yet important portion of secondary structural elements within native proteins. Structural analysis of molecules illustrates 310 helices are usually present at {alpha}-helical extensions, in loops, and as links between beta-strands (34, 35). In fact, 310 helices frequently occur at the termini of regular {alpha}-helices and are even thought to be interchangeable with {alpha}-helices under suitable conditions (34). The 310-helix with {alpha}-helices and beta-strands can form higher order secondary structural motifs in proteins such as glutathione synthetase, myoglobin, and glycogen phosphorylase that can play a role in structural stability, protein folding, and function (35, 36). In fact, the 310-helix located at the carboxyl termini of human {alpha}-lactalbumin is critical to maintain its native structure (37). Other examples that invoke the functionality of a 310-helix include the RNA binding site of the UP1 protein (38) and the catalytic site of p57Kip2 (39). The 310-helix has also been identified in viral proteins, human immunodeficiency virus capsid protein (40), SARS coronavirus replicase polyprotein nsP7 (41), the hepatitis C protease NS3 (42), and the gp41 epitope of human immunodeficiency virus (43), where they have been implicated as potential sites for protein-protein interactions. 310 helices are dynamic structures and its dynamic nature is consistent with its potential functional role as the degradation domain of US2. We propose that the relaxed state of the US2 cytoplasmic tail is stabilized upon the interaction of US2 with class I allowing the tail to interact with the dislocation machinery. Alternatively, the cytoplasmic tail is normally structured in a310-helix and may shift slightly in conformation upon association with class I molecules. In either case, the 310-helix of the tail is utilized as the structural element to promote protein-protein interaction to trigger class I extraction from the ER.


Figure 7
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  		FIGURE 7.
Model of US2-mediated destruction of class I molecules. A, US2 interacts with class I molecules via its lumenal domain and induces a conformational change of the US2 cytoplasmic tail. The US2 tail then engages the dislocation machinery and triggers class I for dislocation. B, the US2 cytoplasmic tail is predicted to be a 310-helix with critical residues on one surface of the helix. The Insight II (Accelrys, San Diego, CA) molecular modeling system was used to model the 14-amino acid US2 cytoplasmic tail in a 310 helical configuration.

 
What type of polypeptides does US2 interact with to trigger class I degradation? Candidates would include ER membrane and cytoplasmic proteins. It is most likely that the initial steps of class I heavy chain degradation (dislocation) involves membrane complexes. These proteins can be the dislocation machinery itself or ubiquitin ligases that would initiate the extraction of class I heavy chains. The association of the tail with the machinery is required for class I degradation, however, the transmembrane of US2 also plays a role in class I destruction. US2 transmembrane domain mutants associate with MHC class I molecules but do not trigger class I destruction.4 These results can only imply that a membrane protein is involved in the destruction of class I molecules. These mutants are essential reagents to identify the polypeptides involved in class I destruction, and most importantly help in defining the initial steps of the dislocation process.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant AI060905. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 Pre-doctoral trainee and supported by United States Public Health Service Institutional Research Training Award AI07647. Back

2 To whom correspondence should be addressed: One Gustave L. Levy Place, Box 1124, New York, NY 10029. Tel.: 212-241-5447; Fax: 212-241-7336; E-mail: Domenico.Tortorella{at}mssm.edu.

3 The abbreviations used are: MHC, major histocompatibility; aa, amino acid(s); ER, endoplasmic reticulum; HCMV, human cytomegalovirus; Ig, immunoglobulin; US, unique short; HA, hemagglutinin. Back

4 K. Oresic, V. Noriega, and D. Tortorella, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank B. Baker, C. Ng, M. Poliskin, and L. Tortorella for critical evaluation of the manuscript. We express gratitude to Dr. M. Mezei (MSSM) for help in modeling the US2 cytoplasmic tail into a 310-helix. We especially thank Dr. H. Ploegh (MIT) for the valuable reagents used in these studies.



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 DISCUSSION
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