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.     

SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein

Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.     

Productive measles virus brain infection and apoptosis in CD46 transgenic mice

Measles virus (MV) infection causes acute childhood disease, associated in certain cases with infection of the central nervous system (CNS) and development of neurological disease. To develop a murine model of MV-induced pathology, we generated several lines of transgenic mice ubiquitously expressing as the MV receptor a human CD46 molecule with either a Cyt1 or Cyt2 cytoplasmic tail. All transgenic lines expressed CD46 protein in the brain. Newborn transgenic mice, in contrast to nontransgenic controls, were highly sensitive to intracerebral infection by the MV Edmonston strain. Signs of clinical illness (lack of mobility, tremors, and weight loss) appeared within 5 to 7 days after infection, followed by seizures, paralysis, and death of the infected animals. Virus replication was detected in neurons from infected mice, and virus was reproducibly isolated from transgenic brain tissue. MV-induced apoptosis observed in different brain regions preceded the death of infected animals. Similar results were obtained with mice expressing either a Cyt1 or Cyt2 cytoplasmic tail, demonstrating the ability of different isoforms of CD46 to function as MV receptors in vivo. In addition, maternally transferred immunity delayed death of offspring given a lethal dose of MV. These results document a novel CD46 transgenic murine model where MV neuronal infection is associated with the production of infectious virus, similarly to progressive infectious measles encephalitis seen in immunocompromised patients, and provide a new means to study pathogenesis of MV infection in the CNS.     

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.     

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.     

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.     

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.     

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.     

Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.     

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.     

Predominant infection of CD150+ lymphocytes and dendritic cells during measles virus infection of macaques

Measles virus (MV) is hypothesized to enter the host by infecting epithelial cells of the respiratory tract, followed by viremia mediated by infected monocytes. However, neither of these cell types express signaling lymphocyte activation molecule (CD150), which has been identified as the receptor for wild-type MV. We have infected rhesus and cynomolgus macaques with a recombinant MV strain expressing enhanced green fluorescent protein (EGFP); thus bringing together the optimal animal model for measles and a virus that can be detected with unprecedented sensitivity. Blood samples and broncho-alveolar lavages were collected every 3 d, and necropsies were performed upon euthanasia 9 or 15 d after infection. EGFP production by MV-infected cells was visualized macroscopically, in both living and sacrificed animals, and microscopically by confocal microscopy and FACS analysis. At the peak of viremia, EGFP fluorescence was detected in skin, respiratory and digestive tract, but most intensely in all lymphoid tissues. B- and T-lymphocytes expressing CD150 were the major target cells for MV infection. Highest percentages (up to 30%) of infected lymphocytes were detected in lymphoid tissues, and the virus preferentially targeted cells with a memory phenotype. Unexpectedly, circulating monocytes did not sustain productive MV infection. In peripheral tissues, large numbers of MV-infected CD11c+ MHC class-II+ myeloid dendritic cells were detected in conjunction with infected T-lymphocytes, suggesting transmission of MV between these cell types. Fluorescent imaging of MV infection in non-human primates demonstrated a crucial role for lymphocytes and dendritic cells in the pathogenesis of measles and measles-associated immunosuppression.     

HIV-1 infection ex vivo accelerates measles virus infection by upregulating signaling lymphocytic activation molecule (SLAM) in CD4+ T cells

Measles virus (MV) infection in children harboring human immunodeficiency virus type 1 (HIV-1) is often fatal, even in the presence of neutralizing antibodies; however, the underlying mechanisms are unclear. Therefore, the aim of the present study was to examine the interaction between HIV-1 and wild-type MV (MVwt) or an MV vaccine strain (MVvac) during dual infection. The results showed that the frequencies of MVwt- and MVvac-infected CD4(+) T cells within the resting peripheral blood mononuclear cells (PBMCs) were increased 3- to 4-fold after HIV-1 infection, and this was associated with a marked upregulation of signaling lymphocytic activation molecule (SLAM) expression on CD4(+) T cells but not on CD8(+) T cells. SLAM upregulation was induced by infection with a replication-competent HIV-1 isolate comprising both the X4 and R5 types and to a lesser extent by a pseudotyped HIV-1 infection. Notably, SLAM upregulation was observed in HIV-infected as well as -uninfected CD4(+) T cells and was abrogated by the removal of HLA-DR(+) cells from the PBMC culture. Furthermore, SLAM upregulation did not occur in uninfected PBMCs cultured together with HIV-infected PBMCs in compartments separated by a permeable membrane, indicating that no soluble factors were involved. Rather, CD4(+) T cell activation mediated through direct contact with dendritic cells via leukocyte function-associated molecule 1 (LFA-1)/intercellular adhesion molecule 1 (ICAM-1) and LFA-3/CD2 was critical. Thus, HIV-1 infection induces a high level of SLAM expression on CD4(+) T cells, which may enhance their susceptibility to MV and exacerbate measles in coinfected individuals.     

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.     

Slow development of measles virus (Edmonston strain) infection in the brain of nude mice

The Edmonston strain of measles virus caused neurologic disease in athymic nude mice by intracerebral inoculation. The incubation periods of the disease, however, were extremely long, ranging from 59 to 140 days when the mice were inoculated with 10(4) plaque forming units (PFU) of the virus. The Edmonston strain was highly infectious in the nude mouse brain since virus infection was established even with 1 PFU of the virus. Virus titers in the brains of infected mice increased with the time of incubation. These results indicate that the extremely long incubation period of the disease is ascribed to very slow development of virus infection in the mouse brain. On the other hand, the incubation periods of the Biken strain of SSPE virus were very short (generally within 2 weeks) even with inoculations of 1 PFU of the virus. However, the extent of the dissemination of infection in brains was not significantly different between the two viruses as examined by immunofluorescent staining.     

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC₅₀) value of each peptide was greater than 1000 μg mL⁻¹. The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL⁻¹ concentration showed a reduction of more than log₁₀ 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log₁₀ 4.8 to log₁₀ 2.5 and log₁₀ 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL⁻¹) to exert its anti-PPRV effect was much less than its CC₅₀ level (>1000 μg mL⁻¹). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.     

Evasion of host defenses by measles virus: wild-type measles virus infection interferes with induction of Alpha/Beta interferon production

Measles is a highly contagious disease currently responsible for over one million childhood deaths, particularly in the developing world. Since alpha/beta interferons (IFNs) are pivotal players both in nonspecific antiviral immunity and in specific cellular responses, their induction or suppression by measles virus (MV) could influence the outcome of a viral infection. In this study we compare the IFN induction and sensitivity of laboratory-passaged attenuated MV strains Edmonston and Moraten with those of recent wild-type viruses isolated and passaged solely on human peripheral blood mononuclear cells (PBMC) or on the B958 marmoset B-cell line. We report that two PBMC-grown wild-type measles isolates and two B958-grown strains of MV induce 10- to 80-fold-lower production of IFN by phytohemagglutinin-stimulated peripheral blood lymphocytes (PBL) compared to Edmonston and Moraten strains of measles. Preinfection of PBL with these non-IFN-inducing MV isolates prevents Edmonston-induced but not double-stranded-RNA-induced IFN production. This suggests that the wild-type viruses can actively inhibit Edmonston-induced IFN synthesis and that this is not occurring by double-stranded RNA. Furthermore, the wild-type MV is more sensitive than Edmonston MV to the effect of IFN. MV is thus able to suppress the synthesis of the earliest mediator of antiviral immunity, IFN-alpha/beta. This could have important implications in the virulence and spread of MV.     

Roles of macrophages in measles virus infection of genetically modified mice

Knowledge of the mechanisms of virus dissemination in acute measles is cursory, but cells of the monocyte/macrophage (MM) lineage appear to be early targets. We characterized the dissemination of the Edmonston B vaccine strain of measles virus (MV-Ed) in peripheral blood mononuclear cells (PBMC) of two mouse strains expressing the human MV-Ed receptor CD46 with human-like tissue specificity and efficiency. In one strain the alpha/beta interferon receptor is defective, allowing for efficient MV-Ed systemic spread. In both mouse strains the PBMC most efficiently infected were F4/80-positive MMs, regardless of the inoculation route used. Circulating B lymphocytes and CD4-positive T lymphocytes were infected at lower levels, but no infected CD8-positive T lymphocytes were detected. To elucidate the roles of MMs in infection, we depleted these cells by clodronate liposome treatment in vivo. MV-Ed infection of splenic MM-depleted mice caused strong activation and infection of splenic dendritic cells (DC), followed by enhanced virus replication in the spleen. Similarly, depletion of lung macrophages resulted in strong activation and infection of lung DC. Thus, in MV infections of genetically modified mice, blood monocytes and tissue macrophages provide functions beneficial for both the virus and the host: they support virus replication early after infection, but they also contribute to protecting other immune cells from infection. Human MM may have similar roles in acute measles.