Fatal Measles Virus Infection Prevented by Brain-Penetrant Fusion Inhibitors

Measles virus (MV) infection causes an acute childhood disease that can include infection of the central nervous system and can rarely progress to severe neurological disease for which there is no specific treatment. We generated potent antiviral peptide inhibitors of MV entry and spreading and MV-induced cell fusion. Dimers of MV-specific peptides derived from the C-terminal heptad repeat region of the MV fusion protein, conjugated to cholesterol, efficiently protect SLAM transgenic mice from fatal MV infection. Fusion inhibitors hold promise for the prophylaxis of MV infection in unvaccinated and immunocompromised people, as well as potential for the treatment of grave neurological complications of measles.     

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.     

Immune Response-Mediated Protection of Adult but Not Neonatal Mice from Neuron-Restricted Measles Virus Infection and Central Nervous System Disease

In many cases of neurological disease associated with viral infection, such as measles virus (MV)-induced subacute sclerosing panencephalitis in children, it is unclear whether the virus or the antiviral immune response within the brain is the cause of disease. MV inoculation of transgenic mice expressing the human MV receptor, CD46, exclusively in neurons resulted in neuronal infection and fatal encephalitis within 2 weeks in neonates, while mice older than 3 weeks of age were resistant to both infection and disease. At all ages, T lymphocytes infiltrated the brain in response to inoculation. To determine the role of lymphocytes in disease progression, CD46⁺ mice were back-crossed to T- and B-cell-deficient RAG-2 knockout mice. The lymphocyte deficiency did not affect the outcome of disease in neonates, but adult CD46⁺ RAG-2⁻ mice were much more susceptible to both neuronal infection and central nervous system disease than their immunocompetent littermates. These results indicate that CD46-dependent MV infection of neurons, rather than the antiviral immune response in the brain, produces neurological disease in this model system and that immunocompetent adult mice, but not immunologically compromised or immature mice, are protected from infection.     

A human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a SLAM- and CD46-independent mechanism

Measles virus (MV) propagates mainly in lymphoid organs throughout the body and produces syncytia by using signaling lymphocyte activation molecule (SLAM) as a receptor. MV also spreads in SLAM-negative epithelial tissues by unknown mechanisms. Ubiquitously expressed CD46 functions as another receptor for vaccine strains of MV but not for wild-type strains. We here show that MV grows and produces syncytia efficiently in a human lung adenocarcinoma cell line via a SLAM- and CD46-independent mechanism using a novel receptor-binding site on the hemagglutinin protein. This infection model could advance our understanding of MV infection of SLAM-negative epithelial cells and tissues.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Cellular tropism and adaptation of the measles virus

Measles virus (MV) is a member of the genus Morbillivirus in the family Paramyxoviridae. Clinical isolates of MV use signaling lymphocyte activating molecule (SLAM) as a cellular receptor. SLAM is mainly expressed on immune cells such as immature thymocytes, activated lymphocytes and mature dendritic cells. This distribution of SLAM can account for the lymphotropism of MV. On the other hand, laboratory strains of MV use CD46 as an alternative receptor, through amino acid change(s) in the receptor binding hemagglutinin protein. Recently, several reports imply the existence of the cellular receptor(s) other than SLAM and CD46. In this review, we discuss the receptor usage of MV and its adaptation to cultured cells.     

Measles virus infects and suppresses proliferation of T lymphocytes from transgenic mice bearing human signaling lymphocytic activation molecule

Humans are the only natural reservoir of measles virus (MV), one of the most contagious viruses known. MV infection and the profound immunosuppression it causes are currently responsible for nearly one million deaths annually. Human signaling lymphocytic activation molecule (hSLAM) was identified as a receptor for wild-type MV as well as for MV strains prepared as vaccines. To better evaluate the role of hSLAM in MV pathogenesis and MV-induced immunosuppression, we created transgenic (tg) mice that expressed the hSLAM molecule under the control of the lck proximal promoter. hSLAM was expressed on CD4(+) and CD8(+) T cells in the blood and spleen and also on CD4(+), CD8(+), CD4(+) CD8(+), and CD4(-) CD8(-) thymocytes. Wild-type MV, after limited passage on B95-8 marmoset B cells, and the Edmonston laboratory strain of MV infected hSLAM-expressing cells. There was a direct correlation between the amount of hSLAM expressed on the cells' surface and the degree of viral infection. Additionally, MV infection induced downregulation of receptor hSLAM and inhibited cell division and proliferation of hSLAM(+) but not hSLAM(-) T cells. Therefore, these tg mice provide the opportunity for analyzing and comparing MV-T cell interactions and MV pathogenesis in cells expressing only the hSLAM MV receptor with those of tg mice whose T cells selectively express another MV receptor, CD46.     

Use of SLAM and PVRL4 and Identification of Pro-HB-EGF as Cell Entry Receptors for Wild Type Phocine Distemper Virus

Signalling lymphocyte activation molecule (SLAM) has been identified as an immune cell receptor for the morbilliviruses, measles (MV), canine distemper (CDV), rinderpest and peste des petits ruminants (PPRV) viruses, while CD46 is a receptor for vaccine strains of MV. More recently poliovirus like receptor 4 (PVRL4), also known as nectin 4, has been identified as a receptor for MV, CDV and PPRV on the basolateral surface of polarised epithelial cells. PVRL4 is also up-regulated by MV in human brain endothelial cells. Utilisation of PVRL4 as a receptor by phocine distemper virus (PDV) remains to be demonstrated as well as confirmation of use of SLAM. We have observed that unlike wild type (wt) MV or wtCDV, wtPDV strains replicate in African green monkey kidney Vero cells without prior adaptation, suggesting the use of a further receptor. We therefore examined candidate molecules, glycosaminoglycans (GAG) and the tetraspan proteins, integrin β and the membrane bound form of heparin binding epithelial growth factor (proHB-EGF),for receptor usage by wtPDV in Vero cells. We show that wtPDV replicates in Chinese hamster ovary (CHO) cells expressing SLAM and PVRL4. Similar wtPDV titres are produced in Vero and VeroSLAM cells but more limited fusion occurs in the latter. Infection of Vero cells was not inhibited by anti-CD46 antibody. Removal/disruption of GAG decreased fusion but not the titre of virus. Treatment with anti-integrin β antibody increased rather than decreased infection of Vero cells by wtPDV. However, infection was inhibited by antibody to HB-EGF and the virus replicated in CHO-proHB-EGF cells, indicating use of this molecule as a receptor. Common use of SLAM and PVRL4 by morbilliviruses increases the possibility of cross-species infection. Lack of a requirement for wtPDV adaptation to Vero cells raises the possibility of usage of proHB-EGF as a receptor in vivo but requires further investigation.     

Measles virus infects both polarized epithelial and immune cells by using distinctive receptor-binding sites on its hemagglutinin

Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.     

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.     

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.     

Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection

To establish a small animal model of severe acute respiratory syndrome (SARS), we developed a mouse model of human severe acute respiratory syndrome coronavirus (SARS-CoV) infection by introducing the human gene for angiotensin-converting enzyme 2 (hACE2) (the cellular receptor of SARS-CoV), driven by the mouse ACE2 promoter, into the mouse genome. The hACE2 gene was expressed in lung, heart, kidney, and intestine. We also evaluated the responses of wild-type and transgenic mice to SARS-CoV inoculation. At days 3 and 7 postinoculation, SARS-CoV replicated more efficiently in the lungs of transgenic mice than in those of wild-type mice. In addition, transgenic mice had more severe pulmonary lesions, including interstitial hyperemia and hemorrhage, monocytic and lymphocytic infiltration, protein exudation, and alveolar epithelial cell proliferation and desquamation. Other pathologic changes, including vasculitis, degeneration, and necrosis, were found in the extrapulmonary organs of transgenic mice, and viral antigen was found in brain. Therefore, transgenic mice were more susceptible to SARS-CoV than were wild-type mice, and susceptibility was associated with severe pathologic changes that resembled human SARS infection. These mice will be valuable for testing potential vaccine and antiviral drug therapies and for furthering our understanding of SARS pathogenesis.     

Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein.     

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.