Dynamic interaction of the measles virus hemagglutinin with its receptor signaling lymphocytic activation molecule (SLAM, CD150)

The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.     

Distinct kinetics for binding of the CD46 and SLAM receptors to overlapping sites in the measles virus hemagglutinin protein

Measles virus (MV) is a human pathogen using two distinct cell surface receptors for entry into host cells. We present here a comparative analysis for binding of the MV receptors CD46 and SLAM to the measles virus hemagglutinin protein (MVH, Edmonston strain). Soluble monomeric and dimeric MVH variants were prepared in mammalian cells and their conformation assessed using a panel of monoclonal antibodies. The two receptor molecules specifically bound to the MVH protein with distinct binding modes. The association rate (k(a)) for SLAM binding to MVH was very low ( approximately 3000 m(-1)s(-1)), about 20 times lower that the k(a) determined for CD46 binding. However, SLAM bound tighter to the virus protein than CD46, as revealed by a 5-fold lower dissociation rate (k(d), approximately 1.5 x 10(-3) s(-1)). These data suggest that the SLAM receptor binds to a less accessible and more hydrophobic surface on MVH than the CD46 receptor, as illustrated in a binding model. Despite the differences in kinetics, receptor competition binding experiments revealed that they recognize overlapping sites in MVH. Indeed, a panel of anti-MVH monoclonal antibodies equally inhibited binding of both receptor molecules. The similar immune reactivity of the two receptor binding sites suggests that the shift in receptor usage by MV may not be driven by immune responses.     

Characterization of a region of the measles virus hemagglutinin sufficient for its dimerization

Attachment of measles virus (MV) to its cellular receptor is mediated by the viral envelope glycoprotein hemagglutinin (H). H exists at the viral surface as a disulfide-linked dimer which may associate into a tetramer. We aimed to define regions of H essential for its homo-oligomerization. To delineate these more precisely, we have generated a series of H ectodomain truncation mutants and studied their abilities to form both homotypic complexes and heterotypic complexes with full-length H. We define a "minimal unit" which is sufficient for MV H dimerization as that encompassing residues 1 to 151. This unit forms both homodimers and heterodimers with full-length H protein, although neither is transported to the cell surface even in the presence of other MV proteins. We show that cysteine residues at positions 139 and 154 are both critical in mediating covalent dimerization, not only of the truncated H mutants but also of full-length MV H protein. Even those cysteine mutants unable to form covalent intermolecular interactions are biologically active, mediating the formation of syncytia, albeit at a reduced rate. We demonstrate that this impaired capacity to mediate cell-to-cell fusion is based mainly on a reduced transport rate of the mutant molecules to the cell surface, indicating a role for covalent intermolecular interactions in efficient transport of MV H dimers to the cell surface.     

Interactions among measles virus hemagglutinin, fusion protein and cell receptor signaling lymphocyte activation molecule (SLAM) indicating a new fusion-trimer model

For measles viruses, fusion on the cell membrane is an important initial step in the entry into the infected cells. The recent research indicated that hemagglutinin firstly leads the conformational changes in the fusion protein then co-mediates the membrane fusion. In the work, we use the co-immunoprecipitation and pull-down techniques to identify the interactions among fusion protein, hemagglutinin and signaling lymphocyte activation molecule (SLAM), which reveal that the three proteins can form a functional complex to mediate the SLAM-dependent fusion. Moreover, under the confocal microscope, fusion protein and hemagglutinin protein can show the cocapping mediated by the SLAM. So fusion protein not only is involved in the fusion but also might directly interact with the SLAM to be a new fusion-trimer model, which might account for the infection mechanism of measles virus.     

V domain of human SLAM (CDw150) is essential for its function as a measles virus receptor

Human signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been shown to be a cellular receptor for measles virus (MV). Chinese hamster ovary cells transfected with a mouse SLAM cDNA were not susceptible to MV and the vesicular stomatitis virus pseudotype bearing MV envelope proteins alone, indicating that mouse SLAM cannot act as an MV receptor. To determine the functional domain of the receptor, we tested the abilities of several chimeric SLAM proteins to function as MV receptors. The ectodomain of SLAM comprises the two immunoglobulin superfamily domains (V and C2). Various chimeric transmembrane proteins possessing the V domain of human SLAM were able to act as MV receptors, whereas a chimera consisting of human SLAM containing the mouse V domain instead of the human V domain no longer acted as a receptor. To examine the interaction between SLAM and MV envelope proteins, recombinant soluble forms of SLAM were produced. The soluble molecules possessing the V domain of human SLAM were shown to bind to cells expressing the MV hemagglutinin (H) protein but not to cells expressing the MV fusion protein or irrelevant envelope proteins. These results indicate that the V domain of human SLAM is necessary and sufficient to interact with the MV H protein and allow MV entry.     

Functional and structural interactions between measles virus hemagglutinin and CD46.

We analyzed the roles of the individual measles virus (MV) surface glycoproteins in mediating functional and structural interactions with human CD46, the primary MV receptor. On one cell population, recombinant vaccinia virus vectors were used to produce the MV hemagglutinin (H) and fusion (F) glycoproteins. As fusion partner cells, various cell types were examined, without or with human CD46 (endogenous or recombinant vaccinia virus encoded). Fusion between the two cell populations was monitored by a quantitative reporter gene activation assay and by syncytium formation. MV glycoproteins promoted fusion with primate cells but not with nonprimate cells; recombinant CD46 rendered nonprimate cells competent for MV glycoprotein-mediated fusion. Markedly different fusion specificity was observed for another morbillivirus, canine distemper virus (CDV): recombinant CDV glycoproteins promoted fusion with primate and nonprimate cells independently of CD46. Fusion by the recombinant MV and CDV glycoproteins required coexpression of H plus F in either homologous or heterologous combinations. To assess the role of H versus F in determining the CD46 dependence of MV fusion, we examined the fusion specificities of cells producing heterologous glycoprotein combinations. The specificity of HMV plus FCDV paralleled that observed for the homologous MV glycoproteins: fusion occurred with primate cells but not with nonprimate cells unless they produced recombinant CD46. By contrast, the specificity of HCDV plus FMV paralleled that for the homologous CDV glycoproteins: fusion occurred with either primate or nonprimate cells with no dependence on CD46. Thus, for both MV and CDV, fusion specificity was determined by H. In particular, the results demonstrate a functional interaction between HMV and CD46. Flow cytometry and antibody coprecipitation studies provided a structural correlate to this functional interaction: CD46 formed a molecular complex with HMV but not with FMV or with either CDV glycoprotein. These results highlight the critical role of the H glycoprotein in determining MV specificity for CD46-positive cells.     

Mechanism of CD150 (SLAM) down regulation from the host cell surface by measles virus hemagglutinin protein

Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.     

Predicting receptor functionality of signaling lymphocyte activation molecule for measles virus hemagglutinin by docking simulation

Predicting susceptibility of various species to a virus assists assessment of risk of interspecies transmission. Evaluation of receptor functionality may be useful in screening for susceptibility. In this study, docking simulation was conducted for measles virus hemagglutinin (MV‐H) and immunoglobulin‐like variable domain of signaling lymphocyte activation molecule (SLAM‐V). It was observed that the docking scores for MV‐H and SLAM‐V correlated with the activity of SLAM as an MV receptor. These results suggest that the receptor functionality may be predicted from the docking scores of virion surface proteins and cellular receptor molecules.     

Apoptosis of human peripheral blood mononuclear cells by wild‐type measles virus infection is induced by interaction of hemagglutinin protein and cellular receptor,SLAM via caspase‐dependent pathway

Although MV infection causes lymphopenia and degradation of cell‐mediated immunity, the mechanisms are poorly known. MV interacts with cellular receptors which mediate virus binding and uptake and are on the surface of PBMC. In this study, apoptosis of MV‐infected PBMC in vitro was analyzed. Both PBMC treated with UV‐inactivated viruses and those infected with live MV underwent apoptosis. Apoptosis of wild‐type MV‐infected PBMC was blocked by anti‐SLAM and anti‐MV hemagglutinin antibodies, respectively. Furthermore, addition of soluble MV hemagglutinin recombinant protein induced apoptosis in PBMC. These data suggest that induction of apoptosis in MV‐infected PBMC is triggered by interaction between hemagglutinin protein of MV and receptor, without other viral components. To further determine the mechanisms of apoptosis, caspase activity was analyzed by Western blotting. Wild‐type virus Yonekawa strain‐induced apoptosis was blocked by pretreatment with pan‐caspase inhibitor (Z‐VAD‐fmk). Intriguingly, the laboratory‐adapted Nagahata strain‐induced apoptosis was not blocked by Z‐VAD‐fmk, indicating that there may be different apoptosis pathways which depend on the viral receptors, SLAM and CD46. Both extrinsic and intrinsic apoptotic pathways, including activation of caspase‐3, ‐8 and ‐9, are involved in Yonekawa strain‐induced apoptosis. Taken together, the findings of this study could open up a new avenue for understanding the molecular mechanisms of MV‐induced PBMC apoptosis and immunosuppression.     

Characterization of a region involved in binding of measles virus H protein and its receptor SLAM (CD150)

Signaling lymphocyte activation molecule (SLAM; also known as CD150) is a newly identified cellular receptor for measles virus (MV). MV Hemagglutinin protein (H) mediates MV entry into host cells by specifically binding to SLAM. Amino acid 27-135 of SLAM was previously shown to be the functional domain to interact with H and used to screen a 10-mer phage display peptide library in this study. After four rounds of screening and sequence analysis, the deduced amino acid sequence of screened peptides SGFDPLITHA and SDWDPLFTHK showed to be highly homologous with amino acid 429-438 of MV H (SGFGPLITHG). Peptides SGFDPLITHA and SDWDPLFTHK specifically inhibited binding of H to SLAM and further inhibition of MV infection suggests that these peptides can be developed to MV blocking reagents and amino acid 429-438 in H protein is functionally involved in receptor binding and may constitute part of the receptor-binding determinants on H protein.     

Clinical isolates of measles virus use CD46 as a cellular receptor

Laboratory strains of measles viruses (MV), such as Edmonston and Halle, use the complement regulatory protein CD46 as a cell surface receptor. The receptor usage of clinical isolates of MV, however, remains unclear. Receptor usage by primary patient isolates of MV was compared to isolates that had been passaged on a variety of tissue culture cell lines. All of the isolates could infect cells in a CD46-dependent manner, but their tropism was restricted according to cell type (e.g., lymphocytes versus fibroblasts). The results indicate that patient isolates that have not been adapted to tissue culture cell lines use CD46 as a receptor. In addition, passaging primary MV patient isolates in B95-8 cells selected variants that had alternate receptor usage compared to the original isolate. Thus, changes in receptor usage by MV are dependent upon the cell type used for isolation. Furthermore, our results confirm the relevance of the CD46 receptor to natural measles infection.     

Identification of amino acid residues involved in the interaction between measles virus Haemagglutin (MVH) and its human cell receptor (signaling lymphocyte activation molecule, SLAM)

Signaling lymphocyte activation molecule (SLAM; also known as CD150) is a newly identified cellular receptor for measles virus (MV). The interaction between MV Haemagglutin (MVH) and SLAM is an initial step for MV entry. We have identified several novel SLAM binding sites at residues S429, T436 and H437 of MVH protein and MVH mutants in these residues dramatically decrease the ability to interaction with the cell surface SLAM and fail to coprecipitation with SLAM in vivo as well as malfunction in syncytium formation. At the same time, K58, S59 and H61 of SLAM was also identified to be critical for MVH and SLAM binding. Further, these residues may be useful targets for the development of measles therapy.     

Antigen mimicry involving measles virus hemagglutinin and human respiratory syncytial virus nucleoprotein.

Intergenic antigenic relationships between measles virus and respiratory syncytial (RS) virus-specific structural components were studied by using monoclonal antibodies. Of 75 monoclonal antibodies against these components, only one, an anti-measles virus hemagglutinin monoclonal antibody, cross-reacted. Immunofluorescence analysis of measles virus- and RS virus-infected cells with this monoclonal antibody showed qualitatively different staining patterns which indicated that the antigen involved in cross-reaction was the RS virus nucleoprotein or phosphoprotein. A radioimmunoprecipitation assay showed the antigen to be the nucleoprotein.     

Physical association of moesin and CD46 as a receptor complex for measles virus.

Recently, two cellular membrane proteins, the membrane cofactor protein CD46 and the membrane-organizing external spike protein, moesin, have been identified to be functionally associated with measles virus (MV) infectivity of cells. We investigated the functional consequences of binding of monoclonal antibodies to both molecules individually and combined on MV attachment, fusion, and plaque formation and the putative direct physical interaction of moesin and CD46. We found that antibodies to moesin or CD46 separately inhibited MV-cell interactions to a high percentage in the plaque test, by approximately 85 and 75%, respectively. The inhibition by combinations of antibodies was additive at low concentrations and complete at high concentrations. This indicates that similar sites of interaction were blocked by steric hindrance. Furthermore, antimoesin antibodies blocked the infection of CD46-negative mouse cell lines with MV. Chemical cross-linking of cell surface proteins indicated the close proximity of CD46 and moesin in the membrane of human cells, and coimmunoprecipitation of moesin with CD46 suggested their physical interaction. Immunohistochemically by electron microscopy, CD46 and moesin were found to be localized at sites of the cellular membrane where MV particles adsorbed. These data support a model of direct interaction of CD46 and moesin in the cellular membrane and suggest that this complex is functionally involved in the uptake of MV into cells.     

CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition

Measles virus (MV) interacts with cellular receptors on the surface of peripheral blood lymphocytes (PBL) which mediate virus binding and uptake. Simultaneously, the direct contact of the viral glycoproteins with the cell surface induces a negative signal blocking progression to the S phase of the cell cycle, resulting in a pronounced proliferation inhibition. We selected a monoclonal antibody (MAb 5C6) directed to the surface of highly MV-susceptible B cells (B95a), which inhibits binding to and infection of cells with MV wild-type and vaccine strains. By screening a retroviral cDNA library from human splenocytes (ViraPort; Stratagene) with this antibody, we cloned and identified the recognized molecule as signaling lymphocytic activation molecule (SLAM; CD150), which is identical to the MV receptor recently found by H. Tatsuo et al. (Nature 406:893-897, 2000). After infection of cells, and after surface contact with MV envelope proteins, SLAM is downregulated from the cell surface of activated PBL and cell lines. Although anti-SLAM and/or anti-CD46 antibodies block virus binding, they do not interfere with the contact-mediated proliferation inhibition. In addition, the cell-type-specific expression of SLAM does not correlate with the sensitivity of cells for proliferation inhibition. The data indicate that proliferation inhibition induced by MV contact is independent of the presence or absence of the virus-binding receptors SLAM and CD46.     

Nectin 4 is the epithelial cell receptor for measles virus

Measles virus (MV) causes acute respiratory disease, infects lymphocytes and multiple organs, and produces immune suppression leading to secondary infections. In rare instances it can also cause persistent infections in the brain and central nervous system. Vaccine and laboratory-adapted strains of MV use CD46 as a receptor, whereas wild-type strains of MV (wtMV) cannot. Both vaccine and wtMV strains infect lymphocytes, monocytes, and dendritic cells (DCs) using the signaling lymphocyte activation molecule (CD150/SLAM). In addition, MV can infect the airway epithelial cells of the host. Nectin 4 (PVRL4) was recently identified as the epithelial cell receptor for MV. Coupled with recent observations made in MV-infected macaques, this discovery has led to a new paradigm for how the virus accesses the respiratory tract and exits the host. Nectin 4 is also a tumor cell marker which is highly expressed on the apical surface of many adenocarcinoma cell lines, making it a potential target for MV oncolytic therapy.     

Multiple amino acid substitutions in hemagglutinin are necessary for wild-type measles virus to acquire the ability to use receptor CD46 efficiently

Measles virus (MV) possesses two envelope glycoproteins, namely, the receptor-binding hemagglutinin (H) and fusion proteins. Wild-type MV strains isolated in B-lymphoid cell lines use signaling lymphocyte activation molecule (SLAM), but not CD46, as a cellular receptor, whereas MV vaccine strains of the Edmonston lineage use both SLAM and CD46 as receptors. Studies have shown that the residue at position 481 of the H protein is critical in determining the use of CD46 as a receptor. However, the wild-type IC-B strain with a single N481Y substitution in the H protein utilizes CD46 rather inefficiently. In this study, a number of chimeric and mutant H proteins, and recombinant viruses harboring them, were generated to determine which residues of the Edmonston H protein are responsible for its efficient use of CD46. Our results show that three substitutions (N390I and E492G plus N416D or T446S), in addition to N481Y, are necessary for the IC-B H protein to use CD46 efficiently as a receptor. The N390I, N416D, and T446S substitutions are present in the H proteins of all strains of the Edmonston lineage, whereas the E492G substitution is found only in the H protein of the Edmonston tag strain generated from cDNAs. The T484N substitution, found in some of the Edmonston-lineage strains, resulted in a similar effect on the use of CD46 to that caused by the E492G substitution. Thus, multiple residues in the H protein that have not previously been implicated have important roles in the interaction with CD46.