Measles virus (MV) hemagglutinin: evidence that attachment sites for MV receptors SLAM and CD46 overlap on the globular head

Measles virus hemagglutinin (MVH) residues potentially responsible for attachment to the wild-type (wt) MV receptor SLAM (CD150) have been identified and localized on the MVH globular head by reference to a revised hypothetical structural model for MVH (www.pepscan.nl/downloads/measlesH.pdb). We show that the mutation of five charged MVH residues which are conserved among morbillivirus H proteins has major effects on both SLAM downregulation and SLAM-dependent fusion. In the three-dimensional surface representation of the structural model, three of these residues (D505, D507, and R533) align the rim on one side of the cavity on the top surface of the MVH globular head and form the basis of a single continuous site that overlaps with the 546-548-549 CD46 binding site. We show that the overlapping sites fall within the footprint of an anti-MVH monoclonal antibody that neutralizes both wt and laboratory-vaccine MV strains and whose epitope contains R533. Our study does not exclude the possibility that Y481 binds CD46 directly but suggests that the N481Y mutation of wt MVH could influence, at a distance, the conformation of the overlapping sites so that affinity to CD46 increases. The relevance of these results to present concepts of MV receptor usage is discussed, and an explanation is proposed as to why morbillivirus attachment proteins are H, whereas those from the other paramyxoviruses are HN (hemagglutinin-neuraminidase).     

Identification of a second major site for CD46 binding in the hemagglutinin protein from a laboratory strain of measles virus (MV): potential consequences for wild-type MV infection

Natural or wild-type (wt) measles virus (MV) infection in vivo which is restricted to humans and certain monkeys represents an enigma in terms of receptor usage. Although wt MV is known to use the protein SLAM (CD150) as a cell receptor, many human tissues, including respiratory epithelium in which the infection initiates, are SLAM negative. These tissues are CD46 positive, but wt MV strains, unlike vaccinal and laboratory MV strains, are not thought to use CD46 as a receptor. We have identified a novel CD46 binding site at residues S548 and F549, in the hemagglutinin (H) protein from a laboratory MV strain, which is also present in wt H proteins. Our results suggest that although wt MV interacts with SLAM with high affinity, it also possesses the capacity to interact with CD46 with low affinity.     

Expression of SLAM (CDw150) on Sf9 cell surface using recombinant baculovirus mediates measles virus infection in the nonpermissive cells

Signaling lymphocytic activation molecule (SLAM; also known as CDw150) has been reported as the receptor of measles virus (MV) interacting with MV hemagglutinin (MVH). In this study, we developed a baculovirus-derived vector, the Bacmid-egfp, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP) under the control of the promoter of very late polyhedrin gene from Autographa californica multiple nucleopolyhedrovirus (AcMNPV), and employed the recombinant baculovirus to express SLAM in Sf9 (Spodoptera frugiperda) cells and investigate SLAM function. The result showed that the integration of the EGFP expression cassette in the Bac-to-Bac system facilitated research with the system without introducing compromises due to its use. SLAM protein fused to His-tag was expressed in Sf9 cells through the modified Bac-to-Bac system. The expressed SLAM was identified as approximately 46 kDa, and it presented on the cell surface, as revealed by fluorescent immunochemical staining and confocal microscopic analysis. The pull-down assay proved that SLAM protein expressed in this system could interact with MVH protein. After incubating with MV vaccine strain S191, cell fusion was only observed in the Sf9 cells expressing both EGFP and SLAM from recombinant baculovirus rather than those expressing EGFP only from the modified viral vector. Furthermore, MV replicated and induced apoptosis in the Sf9 cells with SLAM expression.     

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.     

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.     

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.     

Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion

Clinical isolates of measles virus (MV) use signaling lymphocyte activation molecule (SLAM) as a cellular receptor, whereas vaccine and laboratory strains may utilize the ubiquitously expressed CD46 as an additional receptor. MVs also infect, albeit inefficiently, SLAM(-) cells, via a SLAM- and CD46-independent pathway. Our previous study with recombinant chimeric viruses revealed that not only the receptor-binding hemagglutinin (H) but also the matrix (M) protein of the Edmonston vaccine strain can confer on an MV clinical isolate the ability to grow well in SLAM(-) Vero cells. Two substitutions (P64S and E89K) in the M protein which are present in many vaccine strains were found to be responsible for the efficient growth of recombinant virus in Vero cells. Here we show that the P64S and E89K substitutions allow a strong interaction of the M protein with the cytoplasmic tail of the H protein, thereby enhancing the assembly of infectious particles in Vero cells. These substitutions, however, are not necessarily advantageous for MVs, as they inhibit SLAM-dependent cell-cell fusion, thus reducing virus growth in SLAM(+) B-lymphoblastoid B95a cells. When the cytoplasmic tail of the H protein is deleted, a virus with an M protein possessing the P64S and E89K substitutions no longer grows well in Vero cells yet causes cell-cell fusion and replicates efficiently in B95a cells. These results reveal a novel mechanism of adaptation and attenuation of MV in which the altered interaction of the M protein with the cytoplasmic tail of the H protein modulates MV growth in different cell types.     

Measles virus infection of SLAM (CD150) knockin mice reproduces tropism and immunosuppression in human infection

The human signaling lymphocyte activation molecule (SLAM, also called CD150), a regulator of antigen-driven T-cell responses and macrophage functions, acts as a cellular receptor for measles virus (MV), and its V domain is necessary and sufficient for receptor function. We report here the generation of SLAM knockin mice in which the V domain of mouse SLAM was replaced by that of human SLAM. The chimeric SLAM had an expected distribution and normal function in the knockin mice. Splenocytes from the SLAM knockin mice permitted the in vitro growth of a virulent MV strain but not that of the Edmonston vaccine strain. Unlike in vitro infection, MV could grow only in SLAM knockin mice that also lacked the type I interferon receptor (IFNAR). After intraperitoneal or intranasal inoculation, MV was detected in the spleen and lymph nodes throughout the body but not in the thymus. Notably, the virus appeared first in the mediastinal lymph node after intranasal inoculation. Splenocytes from MV-infected IFNAR(-/-) SLAM knockin mice showed suppression of proliferative responses to concanavalin A. Thus, MV infection of SLAM knockin mice reproduces lymphotropism and immunosuppression in human infection, serving as a useful small animal model for measles.     

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.     

Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor

Both CD46 and signaling lymphocytic activation molecule (SLAM) have been shown to act as cellular receptors for measles virus (MV). The viruses on throat swabs from nine patients with measles in Japan were titrated on Vero cells stably expressing human SLAM. Samples from all but two patients produced numerous plaques on SLAM-expressing Vero cells, whereas none produced any plaques on Vero cells endogenously expressing CD46. The Edmonston strain of MV, which can use either CD46 or SLAM as a receptor, produced comparable titers on these two types of cells. The results strongly suggest that the viruses in the bodies of measles patients use SLAM but probably not CD46 as a cellular receptor.     

Interaction of measles virus (Hallé strain) with CD46: evidence that a common binding site on CD46 facilitates both CD46 downregulation and MV infection.

CD46 acts as a cellular receptor for vaccine strains of measles virus (MV). The MV/CD46 interaction-mediated by the MV attachment glycoprotein, the hemagglutinin (H)-not only facilitates infection but also induces CD46 downregulation. A conflict of opinion exists as to whether a single MVH binding site on CD46, or two separate sites, facilitates the two phenomena. To investigate this conundrum we first tested and compared a panel of CD46-specific monoclonal antibodies (mAbs) for their capacity to block both processes. One (mAb 13/42) abrogated both MV fusion and CD46 downregulation. Mutation of an amino acid (arg59 in the SCR1 of CD46) essential for the epitope of mAb 13/42 resulted in the abrogation of both CD46 downregulation and viral fusion. This strongly suggests that the same MV binding site on CD46 is responsible for both CD46 downregulation and MV infection.     

Nearby clusters of hemagglutinin residues sustain SLAM-dependent canine distemper virus entry in peripheral blood mononuclear cells

Signaling lymphocytic activation molecule (SLAM, CD150) is the universal morbillivirus receptor. Based on the identification of measles virus (MV) hemagglutinin (H) amino acids supporting human SLAM-dependent cell entry, we mutated canine distemper virus (CDV) H and identified residues necessary for efficient canine SLAM-dependent membrane fusion. These residues are located in two nearby clusters in a new CDV H structural model. To completely abolish SLAM-dependent fusion, combinations of mutations were necessary. We rescued a SLAM-blind recombinant CDV with six mutations that did not infect ferret peripheral blood mononuclear cells while retaining full infectivity in epithelial cells.     

Wild-type measles virus with the hemagglutinin protein of the edmonston vaccine strain retains wild-type tropism in macaques

A major difference between vaccine and wild-type strains of measles virus (MV) in vitro is the wider cell specificity of vaccine strains, resulting from the receptor usage of the hemagglutinin (H) protein. Wild-type H proteins recognize the signaling lymphocyte activation molecule (SLAM) (CD150), which is expressed on certain cells of the immune system, whereas vaccine H proteins recognize CD46, which is ubiquitously expressed on all nucleated human and monkey cells, in addition to SLAM. To examine the effect of the H protein on the tropism and attenuation of MV, we generated enhanced green fluorescent protein (EGFP)-expressing recombinant wild-type MV strains bearing the Edmonston vaccine H protein (MV-EdH) and compared them to EGFP-expressing wild-type MV strains. In vitro, MV-EdH replicated in SLAM(+) as well as CD46(+) cells, including primary cell cultures from cynomolgus monkey tissues, whereas the wild-type MV replicated only in SLAM(+) cells. However, in macaques, both wild-type MV and MV-EdH strains infected lymphoid and respiratory organs, and widespread infection of MV-EdH was not observed. Flow cytometric analysis indicated that SLAM(+) lymphocyte cells were infected preferentially with both strains. Interestingly, EGFP expression of MV-EdH in tissues and lymphocytes was significantly weaker than that of the wild-type MV. Taken together, these results indicate that the CD46-binding activity of the vaccine H protein is important for determining the cell specificity of MV in vitro but not the tropism in vivo. They also suggest that the vaccine H protein attenuates MV growth in vivo.     

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.     

Functional interaction between paramyxovirus fusion and attachment proteins

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share approximately 95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110-114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110-114 region of H is structurally conceivable.     

The morbillivirus receptor SLAM (CD150)

Morbilliviruses are highly contagious pathogens that cause some of the most devastating viral diseases of humans and animals, including measles virus (MV), canine distemper virus (CDV), and rinderpest virus (RPV). They replicate mainly in lymphoid organs throughout the body and cause severe immunosuppression accompanied with lymphopenia. We have recently shown that human, canine, and bovine signaling lymphocyte activation molecules (SLAMs; also known as CD150) act as cellular receptors for MV, CDV, and RPV, respectively. In these three morbilliviruses, all strains examined were shown to use SLAMs of their respective host species, and laboratory strains passaged on SLAM-negative cells were found to use, besides SLAM, alternative receptors, such as human CD46 for the Edmonston strain of MV. The use of SLAM as a receptor may be a property common to most, if not all, of the members of morbilliviruses. Human SLAM is a membrane glycoprotein selectively expressed on the cells of the immune system (immature thymocytes, activated lymphocytes, activated monocytes, and mature dendritic cells) and seems to mediate lymphocyte activation and to control interferon-gamma production. The destruction and/or impairment of infected SLAM-positive cells may be a mechanism for the immunosuppression induced by morbilliviruses, but other mechanisms may be also involved.     

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.     

Two different receptors for wild type measles virus

Measles is a highly contagious acute viral disease characterized by a maculopapular rash. It causes severe and temporary immune suppression and is often accompanied by secondary bacterial infections. In 2000, signaling lymphocyte activation molecule (SLAM) was identified as a receptor for measles virus (MV). Observations that SLAM is expressed on cells of the immune system provided a good explanation for the lymphotropic and immunosuppressive nature of MV. However, molecular mechanisms of highly contagious nature of MV have remained unclear. Previously we have demonstrated that MV has an intrinsic ability to infect polarized epithelial cells by using a receptor other than SLAM. Recently, nectin4, a cellular adhesion junction molecule, was identified as the epithelial cell receptor for MV. Understanding the molecular mechanisms of MV to infect both epithelial and immune cells provides a deep insight into measles pathogenesis.     

The Measles Virus Hemagglutinin β-Propeller Head β4-β5 Hydrophobic Groove Governs Functional Interactions with Nectin-4 and CD46 but Not Those with the Signaling Lymphocytic Activation Molecule

Wild-type measles virus (MV) strains use the signaling lymphocytic activation molecule (SLAM; CD150) and the adherens junction protein nectin-4 (poliovirus receptor-like 4 [PVRL4]) as receptors. Vaccine MV strains have adapted to use ubiquitous membrane cofactor protein (MCP; CD46) in addition. Recently solved cocrystal structures of the MV attachment protein (hemagglutinin [H]) with each receptor indicate that all three bind close to a hydrophobic groove located between blades 4 and 5 (β4-β5 groove) of the H protein β-propeller head. We used this structural information to focus our analysis of the functional footprints of the three receptors on vaccine MV H. We mutagenized this protein and tested the ability of individual mutants to support cell fusion through each receptor. The results highlighted a strong overlap between the functional footprints of nectin-4 and CD46 but not those of SLAM. A soluble form of nectin-4 abolished vaccine MV entry in nectin-4- and CD46-expressing cells but only reduced entry through SLAM. Analyses of the binding kinetics of an H mutant with the three receptors revealed that a single substitution in the β4-β5 groove drastically reduced nectin-4 and CD46 binding while minimally altering SLAM binding. We also generated recombinant viruses and analyzed their infections in cells expressing individual receptors. Introduction of a single substitution into the hydrophobic pocket affected entry through both nectin-4 and CD46 but not through SLAM. Thus, while nectin-4 and CD46 interact functionally with the H protein β4-β5 hydrophobic groove, SLAM merely covers it. This has implications for vaccine and antiviral strategies.