Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.     

Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.     

The human cytomegalovirus US10 gene product delays trafficking of major histocompatibility complex class I molecules

Human cytomegalovirus (HCMV) US10 encodes a glycoprotein that binds to major histocompatibility complex (MHC) class I heavy chains. While expression of US10 delays the normal trafficking of MHC class I molecules out of the endoplasmic reticulum, US10 does not obviously facilitate or inhibit the action of two other HCMV-encoded MHC class I binding proteins, US2 and US11.     

Peptide selection by class I molecules of the major histocompatibility complex

Class I molecules of the major histocompatibility complex (MHC) bind peptides derived from cytoplasmic proteins. Comparison of over 100 such peptides reveals the importance of the carboxy-terminal residue in selective binding. Recent evidence implicates the proteases and transporters of the processing pathway in providing peptides with the correct residues at the carboxyl terminus.     

Structural and functional dissection of human cytomegalovirus US3 in binding major histocompatibility complex class I molecules

The human cytomegalovirus US3, an endoplasmic reticulum (ER)-resident transmembrane glycoprotein, forms a complex with major histocompatibility complex (MHC) class I molecules and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes. To identify which parts of US3 confine the protein to the ER and which parts are responsible for the association with MHC class I molecules, we constructed truncated mutant and chimeric forms in which US3 domains were exchanged with corresponding domains of CD4 and analyzed them for their intracellular localization and the ability to associate with MHC class I molecules. All of the truncated mutant and chimeric proteins containing the luminal domain of US3 were retained in the ER, while replacement of the US3 luminal domain with that of CD4 led to cell surface expression of the chimera. Thus, the luminal domain of US3 was sufficient for ER retention. Immunolocalization of the US3 glycoprotein after nocodazole treatment and the observation that the carbohydrate moiety of the US3 glycoprotein was not modified by Golgi enzymes indicated that the ER localization of US3 involved true retention, without recycling through the Golgi. Unlike the ER retention signal, the ability to associate with MHC class I molecules required the transmembrane domain in addition to the luminal domain of US3. Direct interaction between US3 and MHC class I molecules could be demonstrated after in vitro translation by coimmunoprecipitation. Together, the present data indicate that the properties that allow US3 to be localized in the ER and bind MHC class I molecules are located in different parts of the molecule.     

Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation

Human cytomegalovirus (HCMV) glycoprotein US2 causes degradation of major histocompatibility complex (MHC) class I heavy-chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. In US2-expressing cells, MHC proteins present in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. It appears that US2 binding triggers a normal cellular pathway by which misfolded or aberrant proteins are translocated from the ER to cytoplasmic proteasomes. To better understand how US2 binds MHC proteins and causes their degradation, we constructed a panel of US2 mutants. Mutants truncated from the N terminus as far as residue 40 or from the C terminus to amino acid 140 could bind to class I and class II proteins. Nevertheless, mutants lacking just the cytosolic tail (residues 187 to 199) were unable to cause degradation of both class I and II proteins. Chimeric proteins were constructed in which US2 sequences were replaced with homologous sequences from US3, an HCMV glycoprotein that can also bind to class I and II proteins. One of these US2/US3 chimeras bound to class II but not to class I, and a second bound class I HC better than wild-type US2. Therefore, US2 residues involved in the binding to MHC class I differ subtly from those involved in binding to class II proteins. Moreover, our results demonstrate that the binding of US2 to class I and II proteins is not sufficient to cause degradation of MHC proteins. The cytosolic tail of US2 and certain US2 lumenal sequences, which are not involved in binding to MHC proteins, are required for degradation. Our results are consistent with the hypothesis that US2 couples MHC proteins to components of the ER degradation pathway, enormously increasing the rate of degradation of MHC proteins.     

Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes.

Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance energy transfer (FCET) showed that these class I MHC molecules self-associate in liposome membranes, forming small aggregates even at low surface concentrations. The lateral diffusion coefficient (Dlat) of Fl-HLA-A2 decreases with increasing surface protein concentration over a range of lipid:protein molar ratios (L/P) between 8000:1 and 2000:1. The reduction in Dlat of HLA molecules in DMPC liposomes is found to be sensitive to time and temperature. The rotational correlation time for Er-HLA-A2 in DMPC liposomes at 30 degrees C is 87 +/- 0.8 microseconds, at least 10 times larger than that expected for an HLA monomer. There is also significant quenching of donor (Fl-HLA) fluorescence at 37 degrees C in the presence of acceptor-labeled (sulforhodamine-labeled HLA) protein indicating proximity between HLA molecules even at L/P = 4000:1. FPR and FCET measurements with another membrane glycoprotein, glycophorin, give no evidence for its self-association. HLA aggregation measured by FPR, FCET, and TPA was blocked by beta 2-microglobulin, b2m, added to the liposomes. The aggregation of HLA-A2 molecules is not an artifact of their reconstitution into liposomes. HLA aggregates, defined by FCET, were readily detected on the surface of human lymphoblastoid (JY) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of major histocompatibility complex H-2LD class I molecules on spleen colony-forming units

To test whether the major histocompatibility complex class I genes are involved in the regulation of hemopoiesis, the stem cell activities of BALB/c-H-2dm2 (Dm2) mice, which are defective in the expression of H-2L antigens, have been compared with those of the wild-type, BALB/c-Kh, in in vivo and in vitro stem cell assays. In spleen colony-forming unit assays, Dm2 as hosts consistently supported a smaller number of colonies than did BALB/c-Kh. However, both Dm2 and BALB/c-Kh supported a comparable number of colonies in in vitro granulocyte-macrophage colony-forming unit and erythroid colony-forming unit assays. These observations together suggest that the mutation in Dm2 has not affected the hemopoietic potential of the stem cells but may probably affect the hemopoietic microenvironment for the development of the stem cells.     

Identification of class I major histocompatibility complex encoded molecules in the amphibian Xenopus

Class I-like molecules have been immunoprecipitated from Xenopus leukocytes and erythrocytes with alloantisera directed against major histocompatibility complex (MHC)-linked antigens. The heavy chains, depending on the allele examined, have molecular weights of 40 000–44 000 of which 3000 daltons are asparagine-linked carbohydrates, probably present as one N-linked glycan. The presumed analogue of ₂-microglobulin has a molecular weight of 13 000 and bears no asparagine-linked glycans. Family studies show that the heavy chains are encoded by genes residing in or closely linked to the MHC.Abbreviations used in this paper MHC major histocompatibility complex - CML cell-mediated lympholysis - MLR mixed leukocyte reaction - APBS amphibian phosphate-buffered saline - kd kilodalton - LG Xenopus laevis — Xenopus gilli species hybrids - IEF isoelectric focusing Founded and supported by F. Hoffmann-La Roche & Co., Limited Company, CH-4005 Basel, Switzerland.     

A structural determinant of human cytomegalovirus US2 dictates the down-regulation of class I major histocompatibility molecules

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 US2(195)) 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 a 3(10) helical configuration. Such a secondary structure would predict that one side of the 3(10) helical cytoplasmic tail would interact with the extraction apparatus to facilitate the dislocation and subsequent destruction of class I heavy chains.     

Human cytomegalovirus US3 chimeras containing US2 cytosolic residues acquire major histocompatibility class I and II protein degradation properties

Human cytomegalovirus (HCMV) glycoprotein US2 increases the proteasome-mediated degradation of major histocompatibility complex (MHC) class I heavy chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. US2-initiated degradation of MHC proteins apparently involves the recruitment of cellular proteins that participate in a process known as endoplasmic reticulum (ER)-associated degradation. ER-associated degradation is a normal process by which misfolded proteins are recognized and translocated into the cytoplasm for degradation by proteasomes. It has been demonstrated that truncated forms of US2, especially those lacking the cytoplasmic domain (CT), can bind MHC proteins but do not cause their degradation. To further assess how the US2 CT domain interacts with the cellular components of the ER-associated degradation pathway, we constructed chimeric proteins in which the US2 CT domain or the CT and transmembrane (TM) domains replaced those of the HCMV glycoprotein US3. US3 also binds both class I and II proteins but does not cause their degradation. Remarkably, chimeras containing the US2 CT domain caused the degradation of both MHC class I and II proteins although this degradation was less than that by wild-type US2. Therefore, the US2 CT and TM domains can confer on US3 the capacity to degrade MHC proteins. We also analyzed complexes containing MHC proteins and US2, US3, US11, or US3/US2 chimeras for the presence of cdc48/p97 ATPase, a protein that binds polyubiquitinated proteins and likely functions in the extraction of substrates from the ER membrane before the substrates meet proteasomes. p97 ATPase was present in immunoprecipitates containing US2, US11, and two chimeras that included the US2 CT domain, but not in US3 complexes. Therefore, it appears that the CT domain of US2 participates in recruiting p97 ATPase into ER-associated degradation complexes.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

A human herpesvirus 7 glycoprotein, U21, diverts major histocompatibility complex class I molecules to lysosomes.

All members of the herpesvirus family persist in their host throughout life. In doing so, herpesviruses exploit a surprising number of different strategies to evade the immune system. Human herpesvirus 7 (HHV-7) is a relatively recently discovered member of the herpesvirus family, and little is known about how it escapes immune detection. Here we show that HHV-7 infection results in premature degradation of major histocompatibility complex class I molecules. We identify and characterize a protein from HHV-7, U21, that binds to and diverts properly folded class I molecules to a lysosomal compartment. Thus, U21 is likely to function in the normal course of HHV-7 infection to downregulate surface class I molecules and prevent recognition of infected cells by cytotoxic T lymphocytes.     

Role of the adenovirus E3-19k conserved region in binding major histocompatibility complex class I molecules.

The adenovirus early region 3 glycoprotein E3-19k binds to and down regulates major histocompatibility complex (MHC) class I molecules in infected cells. We previously identified a 20-amino-acid conserved region in E3-19k by comparison of protein sequences from four different adenovirus serotypes. The roles of the E3-19k C-terminal and adjacent conserved regions in the interaction with MHC class I molecules have been examined. A functional class I-binding glycoprotein was expressed from the cloned E3 18.5-kDa open reading frame of adenovirus type 35. Truncations and single-amino-acid mutations in the adenovirus type 35 glycoprotein were created by site-directed in vitro mutagenesis and tested for the ability to associate with MHC class I molecules. Deletion of most of the transmembrane domain and cytoplasmic tail did not affect binding to class I molecules. However, removal of an additional 11 amino acids eliminated binding and changed the conformation of the adjacent conserved region. Separate mutations of residues Asp-107 and Met-110, within the conserved region, severely reduced or eliminated binding. These data indicate that the E3-19k conserved region plays a crucial role in binding to MHC class I molecules.     

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment

Misfolded membrane proteins are rapidly degraded, often shortly after their synthesis and insertion in the endoplasmic reticulum (ER), but the exact location and mechanisms of breakdown remain unclear. We have exploited the requirement of MHC class I molecules for peptide to achieve their correct conformation: peptide can be withheld by introducing a null mutation for the MHC-encoded peptide transporter, TAP. By withholding TAP-dependent peptides, the vast majority of newly synthesized class I molecules fails to leave the endoplasmic reticulum and is degraded. We used mice transgenic for HLA-B27 on a TAP1- deficient background to allow visualization by immunoelectron microscopy of misfolded HLA-B27 molecules in thymic epithelial cells. In such HLA transgenic animals, the TAP mutation can be considered a genetic switch that allows control over the extent of folding of the protein of interest, HLA-B27, while the rate of synthesis of the constituent subunits remains unaltered. In TAP1-deficient, HLA-B27 transgenic animals, HLA-B27 molecules fail to assemble correctly, and do not undergo carbohydrate modifications associated with the Golgi apparatus, such as conversion to Endoglycosidase H resistance, and acquisition of sialic acids. We show that such molecules accumulate in an expanded network of tubular and fenestrated membranes. This compartment has its counterpart in normal thymic epithelial cells, and is identified as an ER-Golgi intermediate. We detect the presence of ubiquitin and ubiquitin-conjugating enzymes in association with this compartment, suggesting a nonlysosomal mode of degradation of its contents.     

The human cytomegalovirus US8 glycoprotein binds to major histocompatibility complex class I products

Human cytomegalovirus US8 is a type I membrane protein that partially colocalizes with cellular endosomal and lysosomal proteins. Although US8 does not have discernible effects on the processing and cell surface distribution of major histocompatibility complex (MHC) class I products, we have demonstrated that US8 binds to MHC class I heavy chains in the endoplasmic reticulum.     

Cross-linking staphylococcal enterotoxin A bound to major histocompatibility complex class I is required for TNF-alpha secretion

The mechanism of how superantigens function to activate cells has been linked to their ability to bind and cross-link the major histocompatibility complex class II (MHCII) molecule. Cells that lack the MHCII molecule also respond to superantigens, however, with much less efficiency. Therefore, the purpose of this study was to confirm that staphylococcal enterotoxin A (SEA) could bind the MHCI molecule and to test the hypothesis that cross-linking SEA bound to MHCII-deficient macrophages would induce a more robust cytokine response than without cross-linking. We used a capture enzyme-linked immunosorbent assay and an immunprecipitation assay to directly demonstrate that MHCI molecules bind SEA. Directly cross-linking MHCI using monoclonal antibodies or cross-linking bound SEA with an anti-SEA antibody or biotinylated SEA with avidin increased TNF-alpha and IL-6 secretion by MHCII(-/-) macrophages. The induction of a vigorous macrophage cytokine response by SEA/anti-SEA cross-linking of MHCI offers a mechanism to explain how MHCI could play an important role in superantigen-mediated pathogenesis.