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.     

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.     

Placental extravillous cytotrophoblasts persistently express class I major histocompatibility complex molecules after human cytomegalovirus infection

Human cytomegalovirus (HCMV) downregulates the class I major histocompatibility complexes (MHCs), HLA-A and -B, in infected fibroblasts to escape from antigen-specific cytotoxic T lymphocytes. The HCMV genes responsible for the downregulation of MHCs are US2, US3, US6, and US11, which encode type I membrane proteins working at the endoplasmic reticulum (ER). However, it is largely unknown whether HCMV downregulates the class I MHC molecules in placental extravillous cytotrophoblasts (EVT), which express HLA-C, -E, and -G to protect a semiallogenic fetus from maternal natural killer (NK) cells at the fetomaternal interface. Here, we report that differentiated EVT prepared from human first-trimester chorionic villi persistently express class I MHC molecules upon HCMV infection. When these US proteins were expressed in uninfected EVT, they were localized at the ER in the entire cytoplasm. However, subsequent HCMV infection resulted in dissociation of these US proteins from the ER, which relocated toward the cell membrane. In fibroblasts, these US proteins were localized at the ER before and after HCMV infection. These results suggest that the US gene products are not integrated into ER of HCMV-infected EVT and fail to downregulate class I MHC molecules.     

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.     

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.     

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.     

Distinct trafficking pathways mediate Nef-induced and clathrin-dependent major histocompatibility complex class I down-regulation

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.     

Cell surface expression of an in vitro recombinant class II/class I major histocompatibility complex gene product

Chimeric histocompatibility genes encoding the amino-terminal (beta 1) domain of the class II Ak beta polypeptide and the carboxy-terminal (C2, transmembrane, and intracytoplasmic) domains of either the class I H-2Ld or H-2Dd molecules were stably introduced into mouse L cells. Although both were transcribed, only 5' Ak beta/3' H-2Dd transformants had significant cell membrane expression of a 30-40 kd, heterogeneous glycoprotein containing Ak beta 1 and H-2Dd (C2) serological epitopes. These transformants had a unique pattern of reactivity with monoclonal antibodies previously identified as requiring the Ak beta 1 domain for recognition of complete I-A molecules. These results allow new insight into the structural requirements for cell surface expression of proteins and provide unique cellular reagents for the dissection of humoral and cell-mediated recognition of MHC molecules.     

Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.     

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 nonpolymorphic class I gene in the murine major histocompatibility complex

DNA sequence analysis of a class I gene (Q10), which maps to the Qa2,3 locus in the C57BL/10 (H-2b haplotype) mouse, reveals that it is almost identical to a cDNA clone (pH16) isolated from a SWR/J (H-2q haplotype) mouse liver cDNA library. Exon 5, in particular, has an unusual structure such that a polypeptide product is unlikely to be anchored in the cell membrane. Our findings suggest that the two sequences are derived from allelic class I genes, which are nonpolymorphic, in contrast to H-2K allelic sequences from the same mice, and they may encode liver-specific polypeptides of unknown function. Our previous studies indicate that the Q10 gene is a potential donor gene for the generation of mutations at the H-2K locus by inter-gene transfer of genetic information. Thus the lack of polymorphism in class I genes at the Q10 locus implies either that they are not recipients for such exchanges or that selective pressure prevents the accumulation of mutations in genes at this locus.     

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.     

Human cytomegalovirus gene products US3 and US6 down-regulate trophoblast class I MHC molecules

The epidemiological correlation between human CMV (HCMV) infection and spontaneous fetal loss has been suggested, but the underlying mechanism is not well understood. Fetal cytotrophoblasts, which are in direct contact with the maternal immune system in the uterus during pregnancy, do not express HLA-A and HLA-B, but express the nonclassical class I HLA-G and HLA-C. It has been shown that both HLA-G and HLA-C are capable of inhibiting NK-mediated cell lysis. In our present study, using human trophoblast cell lines as well as other cell lines stably transfected with the human class I genes, we have demonstrated that HCMV US3 and US6 down-regulate the cell-surface expression of both HLA-G and HLA-C by two different mechanisms. HCMV US3 physically associates with both trophoblast class I MHC species, retaining them in the endoplasmic reticulum. In contrast, HCMV US6 inhibits peptide transport by TAP and thus specifically the intracellular trafficking of class I molecules. Therefore, these findings suggest for the first time a possible molecular mechanism underlying HCMV-related spontaneous pregnancy loss.     

Mapping of class I DNA sequences within the human major histocompatibility complex

Mutants that had lost expression of alleles of one or more HLA loci were isolated with immunoselection after gamma-irradiation of a human lymphoblastoid cell line LCL 721. DNAs from the mutants were digested with restriction endonucleases and analyzed by Southern blotting using probes for class I HLA genes. Eight polymorphic cut sites for HindIII and PvuII were discovered in class I-associated sequences of LCL 721. Losses of specific fragments generated by restriction enzymes could be associated with losses of specific antigenic expressions and it was possible in this way to assign HLA-A1, HLA-A2, and HLA-B8 to specific DNA fragments. Patterns of gamma-ray-induced segregations of DNA fragments permitted rough linkage alignment of about 30% of the fragments generated by PvuII. The resultant map showed that there are class I HLA genes on the telomeric side of the HLA-A locus. Restriction enzyme site polymorphisms were also examined in a panel of DNAs isolated from peripheral blood lymphocytes (PBLs) of HLA-typed individuals. This panel of PBL DNA complemented the analysis using the HLA deletion mutants.