Human cytomegalovirus (HCMV) replicates in many diverse cell types in vivo, and entry into different cells involves distinct entry mechanisms and different envelope glycoproteins. HCMV glycoprotein gB is thought to act as the virus fusogen, apparently after being triggered by different gH/gL proteins that bind distinct cellular receptors or entry mediators. A trimer of gH/gL/gO is required for entry into all cell types, and entry into fibroblasts involves trimer binding to platelet-derived growth factor receptor alpha (PDGFRα). HCMV entry into biologically relevant epithelial and endothelial cells and monocyte-macrophages also requires a pentamer, gH/gL complexed with UL128, UL130, and UL131, and there is evidence that the pentamer binds unidentified receptors. We screened an epithelial cell cDNA library and identified the cell surface protein CD147, which increased entry of pentamer-expressing HCMV into HeLa cells but not entry of HCMV that lacked the pentamer. A panel of CD147-specific monoclonal antibodies inhibited HCMV entry into epithelial and endothelial cells, but not entry into fibroblasts. shRNA silencing of CD147 in endothelial cells inhibited HCMV entry but not entry into fibroblasts. CD147 colocalized with HCMV particles on cell surfaces and in endosomes. CD147 also promoted cell-cell fusion induced by expression of pentamer and gB in epithelial cells. However, soluble CD147 did not block HCMV entry and trimer and pentamer did not bind directly to CD147, supporting the hypothesis that CD147 acts indirectly through other proteins. CD147 represents the first HCMV entry mediator that specifically functions to promote entry of pentamer-expressing HCMV into epithelial and endothelial cells.

Human cytomegalovirus (HCMV) infects a wide variety of human cell types by different entry pathways that involve distinct envelope glycoprotein complexes that include gH/gL, a trimer complex consisting of gHgL/gO, and a pentamer complex consisting of gH/gL/UL128/UL130/UL131. We characterized the effects of soluble forms of these proteins on HCMV entry. Soluble trimer and pentamer blocked entry of HCMV into epithelial and endothelial cells, whereas soluble gH/gL did not. Trimer inhibited HCMV entry into fibroblast cells, but pentamer and gH/gL did not. Both trimer and pentamer bound to the surfaces of fibroblasts and epithelial cells, whereas gH/gL did not bind to either cell type. Cell surface binding of trimer and pentamer did not involve heparin sulfate moieties. The ability of soluble trimer to block entry of HCMV into epithelial cells did not involve platelet-derived growth factor PDGFRα, which has been reported as a trimer receptor for fibroblasts. Soluble trimer reduced the amount of virus particles that could be adsorbed onto the surface of epithelial cells, whereas soluble pentamer had no effect on virus adsorption. However, soluble pentamer reduced the ability of virus particles to exit from early endosomes into the cytoplasm and then travel to the nucleus. These studies support a model in which both the trimer and pentamer are required for HCMV entry into epithelial and endothelial cells, with trimer interacting with cell surface receptors other than PDGFR and pentamer acting later in the entry pathway to promote egress from endosomes. HCMV infects nearly 80% of the world's population and causes significant morbidity and mortality. The current antiviral agents used to treat HCMV infections are prone to resistance and can be toxic to patients, and there is no current vaccine against HCMV available. The data in this report will lead to a better understanding of how essential HCMV envelope glycoproteins function during infection of biologically important cell types and will have significant implications for understanding HCMV pathogenesis for developing new therapeutics.

Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.

The structure of a ternary complex formed between a T-cell receptor, a major histocompatibility complex (MHC) protein and a viral peptide provides new insights into the cellular immune response. The results provide a molecular basis for understanding the development of T cells and the reactions leading to transplant rejection and autoimmunity.

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The initiation and maintenance of an immune response to pathogens requires the interactions of cells and proteins that together are able to distinguish appropriate non-self targets from the myriadof self-proteins (Janeway and Bottomly, 1994). This discrimination between self and non-self is in part accomplished by three groups of proteins of the immune system that have direct and specific interactions with antigens: antibodies, T cell receptors (TcR) and major histocompatibility complex (MHC) proteins. Antibodies and TcR molecules are clonally expressed by the B and T cells of the immune system, respectively, defining each progenitor cell with a unique specificity for antigen. In these cell types both antibodies and TcR proteins undergo similar recombination events to generate a variable antigen combining site and thus produce a nearly unlimited number of proteins of different specificities. TcR molecules are further selected to recognize antigenic peptides bound to MHC proteins, during a process known as thymic selection, restricting the repertoire of T cells to the recognition of antigens presented by cells that express MHC proteins at their surface. Thymic selection of TcR and the subsequent restricted recognition of peptide-MHC complexes by peripheral T cells provides a fundamental molecular basis for the discrimination of self from non-sell and the regulation of the immune response (Allen, 1994; Nossal, 1994; von Boehmer, 1994). For example, different classes of T cells are used to recognize and kill infected cells (cytotoxic T cells) arid to provide lymphokiries that induce the niajority of soluble antibody responses of B cells (helper T cells). In contrast to the vast combinatorial and clonal diversity of antibodies and TcRs, a small set of MHC molecules is used to recognize a potentially unlimited universe of foreign peptide antigens for antigen presentation to T cells (Germain, 1994). This poses the problem of how each MHC molecule is capable of recognizing enough peptides to insure an immune response to pathogens. In addition, the specificity of the TcR interaction with MHC-peptide complexes is clearly crucial to the problem of self :non-self discrimination, with implications for both protective immunity and auto-immune disease.

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In this study we examined the association of a promiscuous malaria T cell epitope, CS.T3, to different HLA-DR alleles. A large series of singly substituted or truncated variants of CS.T3 was prepared and tested for the ability to be recognised in association with, or to bind to, three distinct HLA-DR alleles (DR1, DRw11, and DRw14(w6)) and three natural variants of HLA-DRw11. We found that although association with the different DR molecules mapped to identical or closely overlapping regions of the peptide, distinct substitutions could drastically influence the capacity of the peptide to interact with one but not another of the three DR molecules tested. Based on analysis of the distribution of residues recognized by T cell clones restricted to the different DR alleles, we suggest that the peptide CS.T3 is not bound, at least for the three DR examined, as an alpha-helix. In addition we tested three subtypes of DRw11 as APC for the CS.T3 analogues and observed that the peptide is most likely bound in the same conformation to the three natural variants of the DRw11 molecule.