Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors were found to be essential.     

Effect of the alterations in the fusion protein of measles virus isolated from brains of patients with subacute sclerosing panencephalitis on syncytium formation

Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell–cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell–cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV.     

Genetic changes that affect the virulence of measles virus in a rhesus macaque model

To identify genetic changes that lead to the attenuation of measles virus (MV), a strain of MV that is pathogenic in rhesus macaques was adapted to grow in Vero cells, Vero/hSLAM cells and, to simulate the process used to derive live attenuated vaccines, in primary chicken embryo fibroblasts (CEF). Comparison of the complete genomic sequences of the pathogenic wild-type (Davis87-wt) and four cell culture-adapted strains derived from it showed complete conservation of sequence in the Vero/hSLAM-passaged virus. Viruses adapted to Vero cells and CEF had predicted amino acid changes in the nucleocapsid protein, phosphoprotein, V protein, C protein, matrix protein, and the cytoplasmic tail of the hemagglutinin protein. All four cell culture-adapted strains, including the Vero/hSLAM cell-passaged virus, were able to productively infect Vero cells, but the peak viral titers differed. The Vero cell-adapted strains were unable to replicate in Chinese Hamster Ovary cells expressing CD46, indicating that they had not adapted to use the CD46 receptor. The Vero/hSLAM cell-passaged virus retained pathogenicity in rhesus macaques as measured by the appearance of a skin rash while the Vero cell-adapted and CEF-adapted strains had lost the ability to cause a rash. There were no significant differences in viral titers in peripheral blood mononuclear cells among monkeys infected with any of the viral stocks tested. These results identify a limited number of genetic changes in the genome of MV that lead to attenuation in vivo.     

An immunocytometric assay based on dengue infection via DC-SIGN permits rapid measurement of anti-dengue neutralizing antibodies

Dengue remains a global public health threat and development of a safe and effective vaccine is a principal public health goal. The primary correlate of immunity is thought to be neutralizing antibodies. Currently, the plaque reduction neutralization test (PRNT) is the gold standard measure of dengue neutralizing antibody responses, but this test is limited by time-consuming performance. In addition, some feel that use of viral strains adapted to grow in Vero or BHK cells may not accurately reflect protective responses. A human cell line transfected to express a putative natural dengue receptor, DC-SIGN (CD209), was used to measure antibody-mediated dengue neutralization. Using neutralizing monoclonal antibodies, immune sera, and laboratory adapted dengue viruses, serotype-specific neutralizing activity was demonstrated similar to that seen in the Vero PRNT. Importantly, serotype-specific neutralizing activity against recently isolated dengue strains with less heterotypic cross-neutralization than laboratory adapted viruses was also demonstrated.     

Measles virus: immunomodulation and cell tropism as pathogenicity determinants

As important determinants of measles virus (MV) pathogenicity, the MV glycoproteins play a key role in conferring the cellular tropism of this virus, but also in modulating the activity of immunocompetent cells. Whereas all MV strains are able to use CD150 (SLAM) for binding and entry into target cells, only certain, mainly vaccine, strains, can use both CD46 and CD150. Both molecules are down-regulated from the cell surface and this is brought about by both infection and contact with the MV H protein of strains that are able to interact with these molecules. Whereas down-regulation of CD46 could be linked to enhanced sensitivity to complement-mediated lysis, and may thus represent an attenuation marker for vaccine strains, pathogenetic consequences of CD150 down-regulation are unknown as yet. Although the role of CD150 is not entirely clear, viruses containing a wild-type strain-derived H protein revealed a particular tropism for human dendritic cells in vitro, and replicated well in secondary lymphatic tissues of cotton rats where they were also able to cause immunosuppression, as documented by an impaired proliferative response of lymphocytes ex vivo. Most likely, inhibition of T cell expansion by these cells is brought about by another activity of the MV glycoprotein complex, namely by disrupting a pathway important for S-phase entry of T cells, by a mere surface contact.     

The hemagglutinin of recent measles virus isolates induces cell fusion in a marmoset cell line, but not in other CD46-positive human and monkey cell lines, when expressed together with the F protein

Summary.  Measles virus (MV) is efficiently isolated from patients with measles by using B95a cells, a marmoset B cell line. Recent wild-type MV strains isolated using B95a cells did not produce cytopathic effects in any of CD46⁺ primate cell lines examined (except B95a cells), nor did they induce downregulation of CD46. Transfection of the hemagglutinin (H) and fusion (F) genes of the Edmonston strain of MV produced syncytia in HeLa, Cos and B95a cells. By contrast, the expression of the H gene from the two wild-type strains, together with the F gene of the Edmonston strain, resulted in syncytium production in B95a cells, but not in HeLa and Cos cells. Cocultivation of Cos cells expressing the wild-type H protein and the Edmonston strain F protein with B95a cells, but not with HeLa, Jurkat or BJAB cells, generated large syncytia. The results suggest that these recent MV isolates may use a molecule other than CD46 as the cellular receptor or require another coreceptor to infect cells. Accepted October 15, 1997 Received July 18, 1997     

A novel amino acid position in hemagglutinin glycoprotein of measles virus is responsible for hemadsorption and CD46 binding

Summary.  Three recent isolates of measles virus Fu, IMA, and SMD obtained by using B95a cells did not exhibit hemadsorption with African green monkey red blood cells (AGM-RBC). After long-term passage in Vero cells, these Vero cell-adapted strains derived from three isolates obtained the activity to agglutinate AGM-RBC. The primary sequences of the hemagglutinin (H protein) and fusion glycoproteins (F protein) from these two types of viruses were compared and revealed that several important amino acid residues in the H protein do not converge. After adaptation, Fu strain has an Asn to Tyr substitution at position 481 and IMA strain has two substitutions – an Asp to Asn at position 14 and a Ser to Gly at position 546, SMD strain also has a Ser to Gly substitution at position 546. Since the sequences of the F protein were identical between both types of viruses, the hemadsorption alteration from negative to positive might be the result of these substitutions. Site-directed mutagenesis of the H genes were performed to confirm that the substitution of Ser Gly at position 546 and Asn → Tyr at position 481 in the H protein were responsible for hemadsorption alteration. Anti-CD46 monoclonal antibody (M75 and M160) study made clear that these two substitutions also governed the MV H protein’s interaction with CD46 receptor. Our results showed that two important amino acid residues in MV H protein govern the binding to CD46 receptor and hemadsorption. In this paper, we reported a novel amino acid residue at position 546 in MV H protein, which was critical for hemadsorption and CD46 binding. Received May 7, 2001 Accepted October 24, 2001     

An immunohistochemical study of the distribution of the measles virus receptors, CD46 and SLAM, in normal human tissues and subacute sclerosing panencephalitis

We have compared the expression of the known measles virus (MV) receptors, membrane cofactor protein (CD46) and the signaling lymphocyte-activation molecule (SLAM), using immunohistochemistry, in a range of normal peripheral tissues (known to be infected by MV) as well as in normal and subacute sclerosing panencephalitis (SSPE) brain. To increase our understanding of how these receptors could be utilized by wild-type or vaccine strains in vivo, the results have been considered with regard to the known route of infection and systemic spread of MV. Strong staining for CD46 was observed in endothelial cells lining blood vessels and in epithelial cells and tissue macrophages in a wide range of peripheral tissues, as well as in Langerhans' and squamous cells in the skin. In lymphoid tissues and blood, subsets of cells were positive for SLAM, in comparison to CD46, which stained all nucleated cell types. Strong CD46 staining was observed on cerebral endothelium throughout the brain and also on ependymal cells lining the ventricles and choroid plexus. Comparatively weaker CD46 staining was observed on subsets of neurons and oligodendrocytes. In SSPE brain sections, the areas distant from lesion sites and negative for MV by immunocytochemistry showed the same distribution for CD46 as in normal brain. However, cells in lesions, positive for MV, were negative for CD46. Normal brain showed no staining for SLAM, and in SSPE brain only subsets of leukocytes in inflammatory infiltrates were positive. None of the cell types most commonly infected by MV show detectable expression of SLAM, whereas CD46 is much more widely expressed and could fulfill a receptor function for some wild-type strains. In the case of wild-type stains, which are unable to use CD46, a further as yet unknown receptor(s) would be necessary to fully explain the pathology of MV infection.     

Measles virus-induced down-regulation of CD46 is associated with enhanced sensitivity to complement-mediated lysis of infected cells

CD46, the major component of the measles virus (MV) receptor complex and a member of the regulators of complement activity (RCA) gene cluster, is down-regulated in MV-infected cells. We investigated whether the reduction of surface CD46 correlates with enhanced sensitivity of lymphoid and monocytic cells to lysis by activated complement. On human U937 cells, acutely or persistently infected with MV-Edmonston (ED) vaccine strain, infection-dependent down-regulation of CD46 confers sensitivity to activated complement, regardless of the pathway of activation and the specificity of the activating antibodies. Interestingly, down-regulation of CD46 alone is sufficient to confer susceptibility of cells to complement lysis despite the continued surface expression of other RCA proteins such as CD35 and CD55. In primary cultures, both peripheral blood lymphocytes and macrophages are efficiently lysed in the presence of complement activated via the alternative pathway after MV infection. In contrast to the MV-ED infection, infection of cells with the lymphotropic MV wild-type strain WTF does not down-regulate CD46. Cells infected with MV-WTF do not exhibit enhanced susceptibility to complement lysis. These data suggest that MV strains similar to WTF that do not down-regulate CD46 may have an enhanced potential for replication and dissemination within the human host, whereas complement-mediated elimination of cells infected with CD46-down-regulating strains of MV, such as ED, may limit the spread of MV infection, and could thus represent an attenuating factor for MV.     

Enhanced MHC class II-restricted presentation of measles virus (MV) hemagglutinin in transgenic mice expressing human MV receptor CD46

This study analyzes the role of the measles virus (MV) receptor, i.e. the human CD46 molecule, in the MHC class II-restricted presentation of MV hemagglutinin (H). We generated transgenic mice ubiquitously expressing CD46, with a similar level of transgene expression on the surface of antigen-presenting cells (APC), i.e. B cells, dendritic cells (DC) and macrophages. APC isolated from transgenic mice and nontransgenic controls were tested for their ability to present MV H to H-specific CD4⁺ I-E ^d -restricted T cell hybridomas. All three populations of APC were capable of presenting MV to T cell hybridomas, DC being the most efficient. Expression of CD46 on B lymphocytes increased MHC class II-dependent presentation of MV H up to 100-fold, while CD46-transgenic DC stimulated H-specific T cell hybridomas up to 10-fold better than nontransgenic DC. Interestingly, expression of CD46 did not change the presentation efficiency of transgenic macrophages, indicating that CD46-dependent enhancement of antigen presentation depends on the nature of the APC. Furthermore, a single injection of UV-inactivated MV particles into CD46-transgenic mice, but not nontransgenic controls, induced generation of MV-specific T lymphocytes and production of anti-H antibodies, suggesting a role for CD46 in the efficient capture of MV in vivo. These results show for the first time that one ubiquitously expressed cell surface receptor, like CD46, could function in receptor-mediated antigen presentation both in vitro and in vivo and its performance depends on the type of APC which expresses it.     

African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.     

Molecular assembly of CD46 with CD9, alpha3-beta1 integrin and protein tyrosine phosphatase SHP-1 in human macrophages through differentiation by GM-CSF

Human CD46, formerly membrane cofactor protein (MCP), binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic viral infection. CD46 on activated macrophages (Mphi) but not intact monocytes is presumed to be the factor responsible for virus-mediated immune modulation including down-regulation of IL-12 production. As CD46 is expressed on both Mphi and monocytes, the molecular mechanisms responsible for these distinct immune responses remain largely unknown. Here, we found that peripheral blood monocytes treated for 5--8 days with GM-CSF (i.e. mature Mphi) acquired the capacity to assemble CD9, alpha3-beta1 integrin and the tyrosine phosphatase SHP-1 with their CD46. Prior to this maturation stage, Mphi expressed sufficient amounts of CD9 and CD46 but showed no such complex formation, and as in intact monocytes MV replication was markedly suppressed. By flow cytometry and confocal microscopy, the complex was found to assemble on the surface in cells treated with approximately 6 days with GM-CSF but not for approximately 2 days. Notably, an alternative MV receptor SLAM CDw150 was neither expressed nor recruited to this complex throughout GM-CSF-mediated Mphi differentiation. These responses and molecular links were not reproduced in the hamster cell line CHO expressing human CD46 although these cells acquired high susceptibility to MV. Based on these observations, MV susceptibility in human myeloid lineages appears not to be as simple as that observed in human CD46-transfected non-myeloid cells. The molecular complex involving CD46 may confer high MV permissiveness leading to immune modulation in Mphi.     

Cytopathic effect, plaque formation, and lysis of Ehrlichia chaffeensis grown on continuous cell lines.

Ehrlichiae are strict intracellular bacterial pathogens that parasitize leukocytes or other blood cells. Only six agents of the tribe Ehrlichieae, namely, Cowdria ruminantium, Neorickettsia helminthoeca, Ehrlichia risticii, Ehrlichia sennetsu, Ehrlichia canis, and Ehrlichia chaffeensis, have been adapted to growth in continuous cell lines. E. chaffeensis, the agent of human ehrlichiosis, has been cultured only in a cell line of canine origin. We adapted purified cell-free E. chaffeensis for growth in human embryonic lung (HEL) fibroblasts (HEL 299), green monkey kidney cells (Vero), and a human cervical epithelioid carcinoma (HeLa) cell line. We observed a cytopathic effect with both Vero cells and HEL cells and plaque formation with cellular lysis when infected Vero cells were cultured in agar. Human fibroblasts are already commonly used for the isolation of viruses, coexiellae, and rickettsiae. Furthermore, the capability of these cells to support the growth of ehrlichiae suggests that they may be useful for primary isolation of ehrlichiae as well. The cytopathic effect produced in Vero or HEL cells offers a very helpful indicator of the infection. Plaque formation in Vero cells is a new phenomenon not yet reported for ehrlichiae and will allow the titration of inocula and clonal purification of this bacterium.     

Measles as a potential oncolytic virus

The use of replicating viruses for cancer therapy is attracting increasing interest. Numerous viruses are now being considered as potential cancer therapeutics, including the vaccine strain of measles virus (MV). The attenuated strain of measles readily lyses transformed cells, whilst replication and lysis are limited in normal human cells. It has a number of features which make it highly suitable for further development as an oncolytic agent, among them stability and a long history of safety in human use. These features are being combined with its ready potential for genetic manipulations to generate recombinant MVs with desirable therapeutic attributes. This review summarises the pre-clinical studies of the oncolytic efficacy of MV to date. Promising developments in MV engineering--such as re-targeting MV entry to specific cell types and enhancing its utility as a therapeutic agent by expression of non-viral proteins--as well as outstanding issues, such as the role of anti-MV immunity, are highlighted.     

Measles virus interacts with human SLAM receptor on dendritic cells to cause immunosuppression

Measles virus (MV) infects dendritic cells (DCs) resulting in immunosuppression. Human DCs express two MV receptors: CD46 and human signaling lymphocyte activation molecule (hSLAM); thus, the role played by either alone is unclear. Because wild-type (wt) MV uses hSLAM receptor preferentially, we dissected the molecular basis of MV-DC interaction and resultant immunosuppression through the hSLAM receptor by creating transgenic (tg) mice expressing hSLAM on DCs. After infection with wt MV, murine splenic DCs expressing hSLAM receptor had less B7-1, B7-2, CD40, MHC class I, and MHC class II molecules on their surfaces and displayed an increased rate of apoptosis when compared to uninfected DCs. Further, MV-infected DCs failed to stimulate allogeneic T cells and inhibited mitogen-dependent T-cell proliferation. Individual expression of human SLAM, interferon alpha/beta receptor, tumor necrosis factor-alpha, and lymphotoxin-alpha or beta from T cells was not required for MV-infected DCs to inhibit the proliferation of T cells.     

Amino acid substitutions at position 481 differently affect the ability of the measles virus hemagglutinin to induce cell fusion in monkey and marmoset cells co-expressing the fusion protein

Summary.  The hemagglutinin (H) protein of the measles virus (MV) Edmonston strain induced cell fusion in Cos (monkey) and B95a (marmoset) cells, when co-expressed with the fusion (F) protein, whereas the H protein of the wild-type KA strain induced fusion in B95a cells, but not in Cos cells. Asparagine residue at position 481 of the KA H protein was replaced by various amino acids through site-directed mutagenesis. Substitution with tyrosine, which was found at position 481 of the Edmonston H protein, enabled the mutant KA H protein (N481Y) to induce cell fusion in Cos cells co-expressing the F protein, which could be completely blocked by anti-CD46 antibody. This mutant, however, did not cause CD46 downregulation, unlike the Edmonston H protein. The other H protein mutants (N481S, N481T, N481D, N481H, N481F) did not produce syncytia in Cos cells. On the other hand, all of the mutants retained the ability to induce cell fusion in B95a cells. Thus, while tyrosine at position 481 was indispensable for the MV H protein’s interaction with CD46, the residue at this position does not appear to be critically involved in the interaction with the receptor for wild-type strains present on B95a cells. Received March 26, 1999 Accepted April 24, 1999     

The cellular receptor for measles virus--elusive no more

The identity of the measles virus receptor has been controversial. Several years ago CD46 was identified as a cellular receptor for the Edmonston strain of measles virus, but most clinical isolates of measles virus, which are most efficiently isolated in the marmoset B cell line B95a, cannot grow in many CD46+ cell lines. Although some researchers attributed it to post-entry block in viral replication, others believed that there is a receptor other than CD46 for wild-type measles viruses. A new study showed that human signalling lymphocytic activation molecule (SLAM; also known as CDw150) is a cellular receptor for measles virus, including the Edmonston strain. SLAM is expressed on lymphocytes and dendritic cells, and plays an important role in lymphocyte activation. The identification of SLAM as a measles virus receptor nicely explains the pathogenesis of measles virus infection.     

Effective primary isolation of wild-type canine distemper virus in MDCK, MV1 Lu and Vero cells without nucleotide sequence changes within the entire haemagglutinin protein gene and in subgenomic sections of the fusion and phospho protein genes

Canine distemper virus (CDV) is an important pathogen of many carnivores. We are developing a field-based model of morbillivirus virulence and pathogenesis through a study of distemper in naturally infected free-ranging raccoons. The isolation of CDV from raccoon tissues is essential for this work. CDV has often been isolated from animals only after co-cultivation of infected tissues with peripheral blood mononuclear cells derived from specific pathogen-free dogs or similar methods. We explored the utility and consequences of a simpler and cheaper alternative: CDV isolation in Vero, MDCK, and MV1 Lu cells. Virus growth was detected first in MDCK cells, whereas viral cytopathic effects were most obvious in Vero cells. CDV growth in MV1 Lu cells was relatively protracted and occurred without the formation of cytopathic effects. In primary CDV isolates, the entire nucleotide sequence of the receptor binding haemagglutinin (H) gene, and subgenomic fusion (F) and phospho (P) protein gene sequences corresponding to nt 5399-5733 and 2132-2563 of CDV reference strain Onderstepoort, respectively, were identical to those in matched infected tissues. Virus isolation confirmed the presence of CDV in instances where RT-PCR failed to detect CDV in infected tissues. Different viral phenotypes and genotypes were detected. The conservation of H gene sequences in primary CDV isolates suggests that MDCK, MV1 Lu, and Vero cells express proper receptors for wild-type CDV.     

Development of a cell culture system susceptible to measles,canine distemper,and rinderpest viruses

Summary Three strains of chick embryo adapted canine distemper virus (Lederle, Wisconsin, and Onderstepoort strains) and chick embryo adapted LA strain of rinderpest virus were easily adapted to an established line of African green monkey kidney cells (Vero cells), which has been routinely employed for the titration of measles virus. By using these Vero cell adapted strains of canine distemper and rinderpest viruses, techniques of infectivity titration and virus neutralization in Vero cells were established. Comparative studies of cytopathology and growth characteristics of canine distemper, rinderpest, and measles viruses indicated that the behavior of the three viruses in Vero cells is almost the same. The Vero cell system was suggested as a suitable host for the comparative study of the serologic and biologic relationships among measles, canine distemper, and rinderpest viruses.