Measles Virus Glycoproteins Research Articles

Proteolytic cleavage of the fusion protein but not membrane fusion is required for measles virus-induced immunosuppression in vitro.

Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F(0) protein by cellular proteases, such as furin, into the F(1)-F(2) subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F(0) proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F(0) proteins was not due to an impaired F(0)-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F(0) protein.

Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes.

Measles virus (MV)-induced immune suppression during acute measles often leads to secondary viral, bacterial and parasitic infections which severely complicate the course of disease. Previously, we have shown that cotton rats are a good animal model to study MV-induced immune suppression, where proliferation inhibition after ex vivo stimulation of cotton rat spleen cells is induced by the viral glycoproteins (fusion and haemagglutinin proteins). We have now tested a variety of putative mechanisms of MV-induced immune suppression in this animal model. Proliferation inhibition is not due to fusion mediated by the MV glycoproteins and subsequent lysis of cells. Other putative mechanisms like classical anergy (unresponsiveness towards IL-2) or apoptosis do not seem to play a role in MV-induced immune suppression. In contrast, it was shown that spleen cells from infected animals preferentially accumulate in the G0/G1 phase and progress more slowly through the cell cycle after mitogen stimulation in comparison to cells from non-infected animals. These data indicate a retardation of the cell cycle which is correlated with proliferation inhibition and might have severe consequences in mounting an effective immune response.

A recombinant measles vaccine virus expressing wild-type glycoproteins: consequences for viral spread and cell tropism.

Wild-type, lymphotropic strains of measles virus (MV) and tissue culture-adapted MV vaccine strains possess different cell tropisms. This observation has led to attempts to identify the viral receptors and to characterize the functions of the MV glycoproteins. We have functionally analyzed the interactions of MV hemagglutinin (H) and fusion (F) proteins of vaccine (Edmonston) and wild-type (WTF) strains in different combinations in transfected cells. Cell-cell fusion occurs when both Edmonston F and H proteins are expressed in HeLa or Vero cells. The expression of WTF glycoproteins in HeLa cells did not result in syncytia, yet they fused efficiently with cells of lymphocytic origin. To further investigate the role of the MV glycoproteins in virus cell entry and also the role of other viral proteins in cell tropism, we generated recombinant vaccine MVs containing one or both glycoproteins from WTF. These viruses were viable and grew similarly in lymphocytic cells. Recombinant viruses expressing the WTFH protein showed a restricted spread in HeLa cells but spread efficiently in Vero cells. Parental WTF remained restricted in both cell types. Therefore, not only differential receptor usage but also other cell-specific factors are important in determining MV cell tropism.

Measles virus-induced immunosuppression in vitro is associated with deregulation of G1 cell cycle control proteins.

Virus-induced immunosuppression is the major cause of the high morbidity/mortality rates associated with acute measles. It has been shown previously that mitogen-dependent proliferation of peripheral blood lymphocytes (PBL) was strongly impaired after contact with the measles virus (MV) glycoproteins F and H expressed on the surface of infected cells, cells transfected with the corresponding expression constructs or UV-inactivated MV (UV-MV). The state of unresponsiveness was not associated with the induction of apoptosis, and a significant proportion of PBL was found to be arrested in the G0/G1 phase of the cell cycle. It is now shown that cell cycle cessation, rather than complete arrest, is induced after MV glycoprotein contact. No obvious role was found for p53 in the induction of this unresponsiveness. With UV-MV as effector, downregulation of p27, an inhibitor of cyclin-dependent kinase (CDK)-cyclin complexes, was significantly delayed after mitogenic stimulation of human PBL. The activities of both CDK4/6-cyclin D and CDK2-cyclin E complexes for phosphorylation of exogenous substrates in vitro were strongly reduced. CDK4, CDK6, cyclins D3 and E and, to a minor extent, CDK2 failed to accumulate at the protein level after mitogenic stimulation in the presence of UV-MV. These data indicate that MV-induced proliferative unresponsiveness of PBL to mitogenic stimulation is associated with a drastic deregulation of the expression of cell cycle genes essential for the G1/S phase transition.

Processing of N-linked oligosaccharides on the measles virus glycoproteins: importance for antigenicity and for production of infectious virus particles

The envelope of measles virus (MV) particles contains two viral glycoproteins, the haemagglutinin (H) and the fusion (F) protein, which together induce the entry of MV into cells. In the present study, we investigated the role of oligosaccharide processing for the function and antigenicity of the MV glycoproteins by means of glycosidase inhibitors. Golgi α-mannosidase inhibitors (1-deoxymannojirimycin and swainsonine) prevented the oligosaccharides on the MV glycoproteins from obtaining Endo H resistance, but that did not appear to influence in vitro MV infections, indicating that conversion of oligosaccharide chains into the complex form was not required for the function of the MV glycoproteins. The α-glucosidase inhibitor castanospermine (CSP) quantitatively reduced the production of infectious MV particles in cells infected with both vaccine strain and wild-type MV. CSP reduced the detection of the MV F protein by certain monoclonal antibodies (MAbs) that appeared to recognize nonlinear epitopes. CSP also inhibited syncytium formation in MV infected cells, but did not affect MV induced CD46 downregulation, suggesting that CSP primarily influenced the F protein. We propose that CSP induces aberrant folding of MV glycoproteins in a manner that influences their function and antigenicity.

Cell cycle arrest rather than apoptosis is associated with measles virus contact-mediated immunosuppression in vitro.

Acute measles is associated with pronounced immunosuppression characterized both by leukopenia and impaired lymphocyte functions. In an earlier study, we found that mitogen-dependent proliferation of uninfected human peripheral blood lymphocytes (PBLs) and spontaneous proliferation of human cell lines of lymphocytic or monocytic origin was impaired after contact with UV-inactivated, measles virus (MV)-infected cells, UV-inactivated MV or with cells transfected with MV glycoproteins (gp) F and H. We now show that mitogen-stimulated PBLs and Jurkat cell clones either highly sensitive or resistant to CD95-induced apoptosis have a similar sensitivity to MV-induced inhibition and do not undergo apoptosis. Moreover, unimpaired mitogen-dependent upregulation of important activation markers, including IL-2R, was observed in PBL cultures after contact with MV-infected, UV-irradiated presenter cells. This indicates that the cells were indeed viable and acquire a state of activation. Less IL-2 was released from PBLs after contact with MV-infected presenter cells when compared with that released after contact with uninfected cells. However, mitogen-induced proliferation of PBLs was not restored by addition of IL-2 under these conditions. It appeared that a higher fraction of mitogen-stimulated PBLs accumulated in the G0/G1 phase of the cell cycle after contact with MV-infected cells. Thus, the mitogen-unresponsiveness of PBLs seen after contact with MV-infected cells is due to cell cycle arrest rather than apoptosis.

Measles virus-induced immune suppression in the cotton rat (Sigmodon hispidus) model depends on viral glycoproteins.

Immune suppression during measles accounts for most of the morbidity and mortality associated with the virus infection. Experimental study of this phenomenon has been hampered by the lack of a suitable animal model. We have used the cotton rat to demonstrate that mitogen-induced proliferation of spleen cells from measles virus-infected animals is impaired. Proliferation inhibition is seen in all lymphocyte subsets and is not dependent on viral replication. Cells which express the viral glycoproteins (hemagglutinin and fusion protein) transiently by transfection induce proliferation inhibition after intraperitoneal inoculation, whereas application of a recombinant measles virus in which measles virus glycoproteins are replaced with the vesicular stomatitis virus G protein does not have an antiproliferative effect. Therefore, in vivo expression of measles virus glycoproteins is sufficient and necessary to induce inhibition of lymphocyte proliferation.

Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulin

Calmodulin, an EF-hand protein, inhibited the fusion between CD4 + human cells and cells stably expressing HIV-1 envelope proteins. Fusion was also inhibited when HIV-1, HIV-2 or SIV envelope glycoproteins were expressed by vaccinia virus (VV) recombinants, but calmodulin did not inhibit syncytia formation induced by measles virus glycoproteins. Calmodulin also inhibited fusion induced by vPE17, a VV-recombinant expressing a truncated form of HIV-1gp160 which lacks the two known calmodulin-binding sites located in the cytoplasmic domain of gp41. The inhibitory activity was specific to calmodulin among the EF-hand proteins. These observations may be important in understanding the mechanism of retroviral envelope glycoprotein-mediated cell fusion. Several possible mechanisms of action are discussed.

Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro.

A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens. In an in vitro system we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs]. We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100. The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved. Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required. We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins was expressed alone. Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population. Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.

Functional and structural interactions between measles virus hemagglutinin and CD46

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

Cell type-specific MxA-mediated inhibition of measles virus transcription in human brain cells.

Measles virus (MV)-specific transcription in human brain cells is characterized by particularly low abundances of the distal mRNAs encoding the MV envelope proteins. Similar transcriptional restrictions of the closely related vesicular stomatitis virus have been observed in mouse fibroblasts constitutively expressing the interferon-inducible MxA protein (P. Staeheli and J. Pavlovic, J. Virol. 65:4498-4501, 1991). We found that MV infection of human brain cells is accompanied by rapid induction and high-level expression of endogenous MxA proteins. After stable transfection of MxA, human glioblastoma cells (U-87-MxA) released 50- to 100-fold less infectious virus and expression of viral proteins was highly restricted. The overall MV-specific transcription levels were reduced by up to 90%, accompanied by low relative frequencies of the distal MV-specific mRNAs. These restrictions were linked to an inhibition of viral RNA synthesis and not to a decreased stability of the viral RNAs. Our results indicate that expression of MxA is associated with transcriptional attenuation of MV in brain cells, thus probably contributing to the establishment of persistent MV central nervous system infections. In addition, the mechanism of MxA-dependent resistance against MV infection, in contrast to that of vesicular stomatitis virus, is cell type specific, because an inhibition of MV glycoprotein synthesis independent of transcriptional alterations was observed in MxA-transfected human monocytes (J. J. Schnorr, S. Schneider-Schaulies, A. Simon-Jödicke, J. Pavlovic, M. A. Horisberger, and V. ter Meulen, J. Virol. 67: 4760-4768, 1993).

The Role of N-Glycosylation in Cell Fusion Induced by a Vaccinia Recombinant Virus Expressing Both Measles Virus Glycoproteins

We have evaluated the effect of inhibitors of N-glycosylation on syncytia formation in HeLa cell infected by a recombinant vaccinia virus expressing the measles virus hemagglutinin (HA) and fusion (F) glycoproteins (VV-HA/F). Inhibitors which block glucose trimming of oligosaccharide side chains of glycoproteins (glucosidase inhibitors) prevented syncytium formation. On the other hand, inhibitors which block trimming of mannose residues (mannosidase inhibitors) did not prevent syncytium formation. The HA and F synthesized in the presence of either glucosidase or mannosidase inhibitors oligomerized and were transported to the cell surface. Concanavalin A, a mannose-specific lectin, inhibited syncytium formation. The inhibitors did not block the cleavage of the F protein, but in fact enhanced it. These studies demonstrate the importance of protein N-glycosylation in the process of membrane fusion and suggest that trimming of glucose residues and the presence of terminal mannose residues are required for VV-HA/F syncytium induction.

Measles virus glycoproteins: studies on the structure and interaction of the haemagglutinin and fusion proteins.

We have investigated the structure and interaction of the measles virus (MV) glycoproteins expressed at the cell membrane. Cross-linking studies with a variety of chemicals stabilized dimeric forms of the haemagglutinin (HA) or fusion (F) proteins, although by sucrose density gradient analysis, oligomers corresponding to tetramers and larger were observed for both proteins. In cells in which both HA and F were expressed at the surface, their close association was shown by cross-linking and co-immunoprecipitation.

Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus

A monoclonal antibody (MCI20.6) which inhibited measles virus (MV) binding to host cells was previously used to characterize a 57- to 67-kDa cell surface glycoprotein as a potential MV receptor. In the present work, this glycoprotein (gp57/67) was immunopurified, and N-terminal amino acid sequencing identified it as human membrane cofactor protein (CD46), a member of the regulators of complement activation gene cluster. Transfection of nonpermissive murine cells with a recombinant expression vector containing CD46 cDNA conferred three major properties expected of cells permissive to MV infection. First, expression of CD46 enabled MV to bind to murine cells. Second, the CD46-expressing murine cells were able to undergo cell-cell fusion when both MV hemagglutinin and MV fusion glycoproteins were expressed after infection with a vaccinia virus recombinant encoding both MV glycoproteins. Third, M12.CD46 murine B cells were able to support MV replication, as shown by production of infectious virus and by cell biosynthesis of viral hemagglutinin after metabolic labeling of infected cells with [35S]methionine. These results show that the human CD46 molecule serves as an MV receptor allowing virus-cell binding, fusion, and viral replication and open new perspectives in the study of MV pathogenesis.

Measles virus haemagglutinin induces down-regulation of gp57/67, a molecule involved in virus binding.

A surface glycoprotein (gp57/67) was previously shown to be involved in measles virus (MV) binding and characterized in our laboratory. Here, we described down-regulation of cell surface gp57/67 after infection with MV. This effect is specific for MV since cells infected with canine distemper virus, closely related to MV, did not down-regulate gp57/67. The decrease in cell surface gp57/67 correlated with expression of MV glycoproteins and more particularly with the expression of MV haemagglutinin (MV-H). Indeed, expression of MV-H after infection with a vaccinia virus recombinant coding for MV-H was necessary and sufficient to induce down-regulation of gp57/67. Kinetics of cell surface expression of MV-H and gp57/67 showed that the degree of down-regulation was correlated with the amount of MV-H expressed by infected cells. Experiments using antibody-prelabelled gp57/67 and indirect immunofluorescence microscopy allowed us to follow the fate of gp57/67 and showed that down-regulation was occurring by rapid internalization of gp57/67 from the cell surface. These results provide additional evidence that the gp57/67 molecule is closely associated with the pathway of MV infection and also reveal a phenomenon which may be related to viral pathogenesis and persistence.

Measles virus inhibits mitogen-induced T cell proliferation but does not directly perturb the T cell activation process inside the cell

Measles virus (MV) inhibits lymphocyte function in patients, as well as in cells infected in vitro. The proliferation of phytohemagglutinin-stimulated T lymphocytes is suppressed by in vitro MV infection, as shown by the diminished incorporation of [ 3H]thymidine into DNA and the reduced frequency of cells in the S phase of the cell cycle, as compared with mock-infected cells. MV infection itself, however, does not completely block DNA synthesis in infected cells, because infected T cells expressing MV antigens on the cell surface, isolated by fluorescence-activated cell sorter, could still proliferate. Northern blot analysis indicated that the expression of genes induced during T cell activation, such as those encoding interleukin 2 (IL-2), c-myc, IL-2 receptor, IL-6, c-myb, and cdc-2, was not significantly suppressed in MV-infected cells, suggesting that MV does not interfere with the T cell activation process. When anti-MV serum or carbobenzoxy-D-Phe-L-Phe-Gly, a synthetic oligopeptide known to inhibit MV-induced fusion, was added 24 hr after infection, the inhibition of T cell proliferation was reversed in a dose-dependent manner. From these results we propose a model for the inhibition of T cell proliferation by MV; MV glycoproteins expressed on the cell surface of infected cells interact with the MV receptor or other molecules on the cell membrane of adjacent T cells, which in turn affects the proliferation of those T cells.

Measles virus: both the haemagglutinin and fusion glycoproteins are required for fusion

Vaccinia-measles recombinant viruses were used to examine the contribution of the individual measles virus glycoproteins in fusion. Although vaccinia virus recombinants expressing either the haemagglutinin or fusion proteins did not induce fusion in the cell lines examined, a double recombinant expressing both measles virus glycoproteins gave extensive syncytia in cells of human and simian origin. No fusion was observed in mouse, hamster or chicken cells. The fusion induced by the double recombinant could be specifically inhibited with either anti-fusion or anti-haemagglutinin monoclonal antibodies.

Humoral immune response elicited in rats by measles viral membrane antigens presented in liposomes and ISCOMs.

The immunogenicity of measles virus glycoproteins presented associated to liposomes or ISCOMs was compared with that of whole virus and solubilized membrane proteins in W/Fu rats. The rats were immunized three times at ten-day intervals with syngeneic peritoneal exudate cells fed in vitro with the various antigen preparations. A strong and persisting antibody response with haemagglutinin inhibitory and neutralization activity was observed in rats immunized with liposomes, ISCOMs, or virus. The responses were very similar despite the lower dose of protein received by rats immunized with ISCOMs (1 microgram protein) or with liposomes (20 micrograms protein). By contrast, injection of peritoneal exudate cells previously fed in vitro with soluble H and F glycoproteins resulted in only a poor and transient response. The sera from rats immunized with virus, liposomes or ISCOMs contained antibodies immunoprecipitating mainly H and F glycoproteins. Despite a strong enrichment in F polypeptides during the preparation of ISCOMs, they induced an equal anti-H and anti-F antibody response.

Mapping of the major histocompatibility complex and viral antigens on the plasma membrane of a measles virus-infected cell

The two measles virus glycoproteins, the hemagglutinin and fusion protein, are expressed on the surfaces of infected cells. Although the two molecules are chemically distinct, they associate on the cell surface, judging from their ability to comigrate (cocap). However, neither is directly complexed with the major histocompatibility (MHC) gene products, HLA-A, -B, -C or -D, on the plasma membrane, based on results from three distinct assays. First, in tests of capping, these viral glycoproteins failed to comigrate with any HLA determinant. Second, electron microscopy showed that the viral glycoproteins occupied domains on the plasma membrane distinct from MHC gene products; 125I labeling of cell surface determinants and subsequent analysis by immune precipitation and PAGE confirmed this result. Third, incubation of measles virus-infected cells in the presence of monoclonal or polyclonal antibodies to measles virus glycoproteins removed the viral glycoproteins from the cells' surfaces but did not cause a corresponding decrease in amounts of HLA molecules. These results indicate that the hemagglutinin and fusion polypeptides of measles virus lie in close association on the plasma membrane; however, neither is linked with MHC gene products.

Mechanisms of killing of measles virus-infected cells by human lymphocytes: Interferon associated and unassociated cell-mediated cytotoxicity

Human fibroblasts infected with measles virus express viral hemagglutinin (HA) and fusion (F) glycoproteins on their surface. These infected cells are killed by peripheral blood lymphocytes (PBLs) within 2 to 4 hr of incubation in vitro. Both syngeneic and allogeneic PBLs harvested from human adults, all previously recovered from childhood measles virus infection (immune donors), are efficient in mediating this cytotoxicity. Cytotoxicity is also expressed by PBLs from a donor with no history of measles virus and lacking circulating anti-measles virus antibodies. This cell-mediated cytotoxicity (CMC) is not due to anti-measles virus IgG in that it is not abrogated by purified F(ab′) 2 fragments from rabbit IgG anti-human F(ab′) 2. The killer lymphocytes are Fc-receptor positive and erythrocyte rosetting and non-erythrocyte resetting, as assessed by both positive and negative selection experiments. Exposure of viral glycoproteins at surfaces of infected cells is necessary for this early CMC, which can be completely abrogated by F(ab′) 2 fragments to measles virus glycoproteins. However, the same F(ab′) 2 fragments are not effective in blocking CMC after an 8-hr incubation period (late CMC), suggesting that a different mechanism of CMC operates at the later time. Interferon (IFN)-like activity cannot be demonstrated in culture fluids within 4 hr of incubation, and early CMC is not inhibited by antibody to human IFN. In contrast, after 8 hr of incubation, culture fluids do possess antiviral activity, and do have the capacity to recruit new CMC indicating that IFN is now present in the system. Therefore, viral glycoproteins expressed at the surface of infected cells induced CMC early in the infectious cycle and this CMC is not associated with IFN-like activity. Later during the infection, a different IFN-associated CMC is also present.