Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

Measles virus-induced immunosuppression.

Immunosuppression is the major cause of infant death associated with acute measles and therefore of substantial clinical importance. Major hallmarks of this generalized modulation of immune functions are (1) lymphopenia, (2) a prolonged cytokine imbalance consistent with suppression of cellular immunity to secondary infections, and (3) silencing of peripheral blood lymphocytes, which cannot expand in response to ex vivo stimulation. Lymphopenia results from depletion, which can occur basically at any stage of lymphocyte development, and evidently, expression of the major MV receptor CD150 plays an important role in targeting these cells. Virus transfer to T cells is thought to be mediated by dendritic cells (DCs), which are considered central to the induction of T cell silencing and functional skewing. As a consequence of MV interaction, viability and functional differentiation of DCs and thereby their expression pattern of co-stimulatory molecules and soluble mediators are modulated. Moreover, MV proteins expressed by these cells actively silence T cells by interfering with signaling pathways essential for T cell activation.

Incorporation and visualization of azido-functionalized N-oleoyl serinol in Jurkat cells, mouse brain astrocytes, 3T3 fibroblasts and human brain microvascular endothelial cells.

The synthesis and biological evaluation of azido-N-oleoyl serinol is reported. It mimicks biofunctional lipid ceramides and has shown to be capable of click reactions for cell membrane imaging in Jurkat and human brain microvascular endothelial cells.

Measles virus replication in neural cells.

Measles virus gene expression is attenuated in neural cells by mechanisms that affect both viral transcription and translation. Host enzymes that hypermutate viral genes, and those induced by cytokines, may act cooperatively to slow viral replication and to favor persistent measles virus infections in the human central nervous system.

Dendritic cells and measles virus infection.

Measles is a major cause of childhood mortality in developing countries which is mainly attributed to the ability of measles virus (MV) to suppress general immune responses. Paradoxically, virus-specific immunity is efficiently induced, which leads to viral clearance from the host and confers long-lasting protection against reinfection. As sensitisers of pathogen encounter and instructors of the adaptive immune response, dendritic cells (DCs) may play a decisive role in the induction and quality of the MV-specific immune activation. The ability of MV wild-type strains in particular to infect DCs in vitro is dearly established, and the receptor binding haemagglutinin protein of these viruses essentially determines this particular tropism. DC maturation as induced early after MV infection is likely to be of crucial importance for the induction of MV-specific immunity. DCs may, however, be instrumental in MV-induced immunosuppression. (1) T cell depletion could be brought about by DC-T cell fusion or TRAIL-mediated induction of apoptosis. (2) Inhibition of stimulated IL-12 production from MV-infected DCs might affect T cell responses in qualitative terms in favouring Th2 and suppressing Th1 responses. (3) The viral glycoprotein complex expressed at high levels on infected DCs late in infection is able to directly inhibit T cell proliferation by surface contact-dependent negative signalling. This most likely accounts for the failure of infected DC cultures to stimulate allogeneic and inhibit mitogen-stimulated T cell proliferation in vitro and the pronounced proliferative unresponsiveness of T cell ex vivo to polyclonal and antigen-specific stimulation which is a central finding of MV-induced immunosuppression.

Measles virus induced immunosuppression: targets and effector mechanisms.

A profound, transient suppression of immune functions during and after the acute infection is the major cause of more than one million cases of infant deaths associated with measles worldwide. Concommittant with the generation of an efficient measles virus (MV) specific immunity, immune responses towards other pathogens are strongly impaired and provide the basis for the establishment and severe course of opportunistic infections. The molecular basis for MV-induced immunosuppression has not been resolved as yet. Similar to other immunosuppressive viruses, MV is lymphotropic and viral nucleic acid and proteins are detectable in peripheral blood mononuclear cells (PBMC). It is considered central to MV-induced immunosuppression that PBMC isolated from patients largely fail to proliferate in response to antigen specific and polyclonal stimulation. The low abundancy of MV-infected PBMC suggests that MV-induced immunosuppression is not directly caused by infection-mediated cell loss or fusion, but rather by indirect mechanisms such as deregulation of cytokines or surface contact-mediated signaling which may lead to apoptosis or impair the proliferative response of uninfected PBMC. Evidence for a role of any of these mechanisms was obtained in vitro, however, much has still to be learned about the tropism of MV and its interactions with particular host cells such as dendritic cells in vivo.

The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures.

To define domains of the human MxA GTPase involved in GTP hydrolysis and antiviral activity, we used two monoclonal antibodies (mAb) directed against different regions of the molecule. mAb 2C12 recognizes an epitope in the central interactive region of MxA, whereas mAb M143 is directed against the N-terminal G domain. mAb 2C12 greatly stimulated MxA GTPase activity, suggesting that antibody-mediated crosslinking enhances GTP hydrolysis. In contrast, monovalent Fab fragments of 2C12 abolished GTPase activity, most likely by blocking intramolecular interactions required for GTPase activation. Interestingly, intact IgG molecules and Fab fragments of 2C12 both prevented association of MxA with viral nucleocapsids and neutralized MxA antiviral activity in vivo. mAb M143 had no effect on MxA function, indicating that this antibody binds outside functional regions. These data demonstrate that the central region recognized by 2C12 is critical for regulation of GTPase activity and viral target recognition.

Pathogenic aspects of measles virus infections.

Measles virus (MV) infections normally cause an acute self limiting disease which is resumed by a virus-specific immune response and leads to the establishment of a lifelong immunity. Complications associated with acute measles can, on rare occasions, involve the central nervous system (CNS). These are postinfectious measles encephalitis which develops soon after infection, and, months to years after the acute disease, measles inclusion body encephalitis (MIBE) and subacute sclerosing panencephalitis (SSPE) which are based on a persistent MV infection of brain cells. Before the advent of HIV, SSPE was the best studied slow viral infection of the CNS, and particular restrictions of MV gene expression as well as MV interactions with neural cells have revealed important insights into the pathogenesis of persistent viral CNS infections. MV CNS complication do, however, not large contribute to the high rate of mortality seen in association with acute measles worldwide. The latter is due to a virus-induced suppression of immune functions which favors the establishment of opportunistic infections. Mechanisms underlying MV-mediated immunosuppression are not well understood. Recent studies have indicated that MV-induced disruption of immune functions may be multifactorial including the interference with cytokine synthesis, the induction of soluble inhibitory factors or apoptosis and negative signalling to T cells by the viral glycoproteins expressed on the surface of infected cells, particularly dendritic cells.

Pathogenetic aspects of measles virus infections.

In this review we briefly summarise virological and immunological aspects of acute and persistent measles virus (MV) infections as found in man and further investigated in animal models and tissue culture. Several mechanisms contributing to MV persistence are discussed. Recent findings, especially the analysis of CD46 and moesin as receptor acting molecules for MV, correlate well with the tropism of MV and open new fields for the investigation of MV associated immunosuppression and central nervous system (CNS) complications.

Differential induction of cytokines by primary and persistent measles virus infections in human glial cells.

The effect of measles virus (MV) infection on mRNA expression and protein synthesis of cytokines in human malignant glioma cell lines (D-54 and U-251) was investigated. Primary MV infections led in both cell lines to the induction of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interferon-beta (IFN-beta), and tumor necrosis factor-alpha (TNF-alpha). In contrast, persistently infected astrocytoma lines continually produced IL-6 (two out of 12 lines high levels) and IFN-beta, whereas only 1 out of 12 lines synthesized TNF-alpha and none IL-1 beta. The pathways for induction of IL-1 beta and TNF-alpha expression were not suppressed by the persistent MV infection, since IL-1 beta and TNF-alpha could be induced by external stimuli like diacylglycerol analog plus calcium ionophore. Interestingly, persistently infected astrocytoma cells synthesized considerably higher levels of IL-1 beta and TNF-alpha than uninfected cells after additional external induction. These results suggest that in the central nervous system (CNS) of SSPE patients a percentage of persistently infected astrocytes may continually synthesize IL-6 and IFN-beta, and in the presence of additional external stimuli, as possibly provided by activated lymphocytes, might overexpress the inflammatory cytokines IL-1 beta and TNF-alpha. This may be of pathogenetic significance in CNS diseases associated with persistent MV infections.

Measles virus-induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface.

Measles virus (MV) infection promotes maturation of dendritic cells (DC), but also interferes with DC functions, and MV renders the DC inhibitory for T cell proliferation. We now describe that MV infection triggers the release of type I IFN from monocyte-derived DC (Mo-DC) which contributes to DC maturation. There is no evidence that soluble mediators are released interfering with the stimulatory activity of uninfected DC. Since inhibition of allogeneic T cell proliferation was unaffected by a fusion inhibitory peptide (Z-fFG), MV infection of T cells did not contribute to inhibition. Allogeneic T cell proliferation depended on the percentage of DC expressing MV F/H glycoproteins within the DC population and their surface expression levels, was induced upon addition of UV-inactivated MV to a mixed lymphocyte reaction stimulated by lipopolysaccharide-matured DC, and was not induced by DC infected with a recombinant MV encoding the ectodomain of vesicular stomatitis virus G protein (MG/FV) instead of the MV glycoproteins. Similarly, DC infected with MV, but not with MG/FV inhibited mitogen-induced proliferation of T cells. Thus, a dominant inhibitory signal is delivered to T cells by the MV glycoproteins on the surface of DC overcoming positive signals by co-stimulatory molecules promoted by maturation factors released from infected DC.

Measles virus interactions with cellular receptors: consequences for viral pathogenesis.

Although CNS complications occurring early and late after acute measles are a serious problem and often fatal, the transient immunosuppression lasting for several weeks after the rash is the major cause of measles-related morbidity and mortality worldwide. This review is focused on the interactions of measles virus (MV) with cellular receptors on neural and lymphoid cells which are important elements in viral pathogenesis. First, the cognate MV receptors, CD46 and CD150, are important components of viral tropism by mediating binding and entry. Second, however, additional unknown cellular surface molecules may (independently of viral uptake) after interaction with the MV glycoprotein complex act as signaling molecules and thereby modulate cellular survival, proliferation, and specific functions.

Expression of the interferon-induced MxA protein in viral encephalitis.

MxA protein accumulates cytoplasmically in response to interferon stimulation, and mediates resistance against several viruses. In order to test whether MxA may serve as a diagnostic tool for viral infections of the central nervous system (CNS), we performed MxA immunohistochemistry on biopsies and autopsies of 57 patients with neurological disorders of known viral and nonviral aetiology. MxA was detectable in all HIV patients with proven opportunistic viral encephalitis, in all patients suffering from isolated viral encephalitis, in one of three HIV patients with cerebral toxoplasmosis, and in one case of micronodular encephalitis. No MxA was detectable in HIV patients with isolated HIV encephalitis or HIV infection accompanied by an opportunistic nonviral disorder. We were unable to show MxA expression in a variety of nonviral inflammatory and noninflammatory disorders of the CNS. Several cases of Rasmussen's encephalitis and multiple sclerosis tested negative, arguing against their possible viral aetiology. Two-colour immunohistochemistry identified macrophages and activated microglia as MxA expressing cells. In all studied cases MxA expression was accompanied by a marked T-cell infiltrate. Therefore, the detection of MxA-protein is a sensitive adjuvant marker for those cases of viral encephalitis which are accompanied by pronounced lymphocytic infiltrates.

Restricted expression of measles virus in primary rat astroglial cells.

Persistent infection of the central nervous system (CNS) with measles virus (MV) is associated with characteristic restrictions of viral envelope gene expression as documented in subacute sclerosing panencephalitis (SSPE), measles inclusion body encephalitis (MIBE), or subacute measles encephalitis (SAME) in rats. To determine whether these restrictions are the result of a long lasting virus-host cell interaction or primarily based on intrinsic brain cell factors MV gene expression was analyzed in primary rat astroglial cultures. It could be shown that MV infection of these cells led to a defective replication cycle with a reduced synthesis of viral envelope proteins and a steep expression gradient of the monocistronic viral mRNAs similar to the findings in brain tissue of SSPE, MIBE, and SAME. This restriction of MV gene expression has not been observed in cells of nonneural origin. We suggest that this cell-type specific regulation of MV gene expression contributes to early events in the establishment of MV persistent infection in CNS tissue.

Spontaneous and differentiation-dependent regulation of measles virus gene expression in human glial cells.

The expression of measles virus (MV) in six different permanent human glioma cell lines (D-54, U-251, U-138, U-105, U-373, and D-32) was analyzed. Although all cell lines were permissive for productive replication of all MV strains tested, U-251, D-54, and D-32 cells spontaneously revealed restrictions of MV transcription similar to those observed for primary rat astroglial cells and brain tissue. In vitro differentiation of D-54 and U-251 cells by substances affecting the intracellular cyclic AMP level caused a significant reduction of the expression of the viral proteins after 18, 72, and 144 h of infection. This pronounced restriction was not paralleled to a comparable level by an inhibition of the synthesis and biological activity in vitro of virus-specific mRNAs as shown by quantitative Northern (RNA) blot analyses and in vitro translation. The block in viral protein synthesis could not be attributed to the induction of type I interferon by any of the substances tested. Our findings indicate that down-regulation of MV gene expression in human brain cells can occur by a cell type-dependent regulation of the viral mRNA transcription and a differentiation-dependent regulation of translation, both of which may be crucial for the establishment of persistent MV infections in the central nervous system.

Antibody-induced restriction of viral gene expression in measles encephalitis in rats.

After infection with the neurotropic CAM/RBH measles virus (MV) strain, newborn Lewis rats succumb to an acute necrotizing encephalopathy. Passive transfer of neutralizing monoclonal antibodies directed against MV hemagglutinin prevented this disease process. Instead, either an antibody-induced acute or subacute measles encephalitis developed after a prolonged incubation period with a restricted expression of MV structural proteins. The molecular biological analysis of MV gene expression in brain tissue of rats treated with MV-neutralizing antibodies revealed a transcriptional restriction of viral mRNAs, particularly for the envelope proteins, leading to a steep expression gradient. Based on in situ hybridization, it was concluded that the efficiency of transcription of viral genes at the single-cell level is reduced compared with that of controls. Passive immunization with monoclonal antibodies directed against other MV structural proteins proved to be ineffective. Similar results were obtained in MV-infected weanling Brown Norway rats. These rats developed a clinically silent encephalitis in the presence of high titers of neutralizing antibodies. In such animals, a pronounced attenuation of the viral gene transcription was observed. These findings indicated that neutralizing antibodies directed against a restricted set of specific antigenic sites on the viral hemagglutinin protein expressed on cell membranes exert a modulating effect on the viral gene expression at the level of transcription. This phenomenon contributes to the switch from the acute cytopathic effect to a persistent infection in the central nervous system.

Measles virus-induced immunosuppression in vitro is independent of complex glycosylation of viral glycoproteins and of hemifusion.

Expression of the measles virus (MV) F/H complex on the surface of viral particles, infected cells, or cells transfected to express these proteins (presenter cells [PC]) is necessary and sufficient to induce proliferative arrest in both human and rodent lymphoid cells (responder cells [RC]). This inhibition was found to occur independent of apoptosis and soluble mediators excluded by a pore size filter of 200 nm released from either PC or RC. We now show that reactive oxygen intermediates which might be released by RC or PC also do not contribute to MV-induced immunosuppression in vitro. Using an inhibitor of Golgi-resident mannosidases (deoxymannojirimycin), we found that complex glycosylation of the F and H proteins is not required for the induction of proliferative arrest of RC. As revealed by our previous studies, proteolytic cleavage of the MV F protein precursor into its F1 and F2 subunits, but not of F/H-mediated cellular fusion, was found to be required, since fusion-inhibitory peptides such as Z-D-Phe-L-Phe-Gly (Z-fFG) did not interfere with the induction of proliferative inhibition. We now show that Z-fFG inhibits cellular fusion at the stage of hemifusion by preventing lipid mixing of the outer membrane layer. These results provide strong evidence for a receptor-mediated signal elicited by the MV F/H complex which can be uncoupled from its fusogenic activity is required for the induction of proliferative arrest of human lymphocytes.

Expression of measles virus RNA in peripheral blood mononuclear cells of patients with measles, SSPE, and autoimmune diseases.

In order to characterize measles virus (MV) infection in peripheral blood mononuclear cells (PBMCs), RNA was isolated from PBMCs after PHA-stimulation for 72 hr of 9 patients with acute measles, 16 patients with subacute sclerosing panencephalitis (SSPE), 13 patients with various autoimmune diseases, and 16 healthy control donors. The RNA obtained was screened for the presence of MV N (nucleocapsid) gene specific transcripts of either positive or negative orientation in a S1 nuclease protection assay. The sensitivity of this assay allowed us to detect one infected cell in 20,000 PBMCs or 0.1 to 0.05 copies of MV-specific RNA per cell. Using single-stranded DNA or RNA probes expression of MV genomic RNA of negative polarity could be detected in only one case of acute measles and one healthy control donor. Conversely, N-specific transcripts of positive polarity, indicating active transcription, could only be detected in patients with acute measles. In addition, in infected PBMCs and in a persistently MV-infected B cell line positive stranded N-specific transcripts containing leader usually present at very low frequency have been found in relatively increased amounts in comparison with transcripts lacking leader. Whereas the ratio of these RNA species during lytic infection with MV in Vero cells is about 1:50, the ratio found here ranges from 1:3 to 1:10. This altered ratio indicates a specific regulation of MV specific transcription in cells of lymphoid origin that has not been found in any other cell system analyzed.

Augmentation of major histocompatibility complex class I and ICAM-1 expression on glial cells following measles virus infection: evidence for the role of type-1 interferon.

An intracellular staining procedure for the cytoskeletal marker, glial fibrillary acidic protein of astrocytes, has been developed which allows flow cytometric phenotyping of astrocytes within complex mixtures of glial cells. Employing this technique, we show here that measles virus infection of rat mixed glial cell cultures results in a rapid augmentation of major histocompatibility complex (MHC) class I and ICAM-1 on the majority of astrocytes in culture. MHC class I levels are increased on macrophages/microglia but ICAM-1 expression is not normally affected on this cell type. Some MHC class II induction is also observed after virus infection but only on astrocytes. A type-I interferon (IFN)-inducible protein, Mx, was identified in cultured glial cells after infection. Qualitatively comparable MHC class I and ICAM-1 enhancement after addition of type-I IFN, supports the conclusion that this cytokine(s) released as a result of virus infection, is responsible for alterations in the expression of molecules on glial cells, that are involved in T cell recognition. Astrocytes after viral infection were more susceptible to alloantigen-specific cytotoxic T lymphocytes and cytotoxic T lymphocyte activity was substantially reduced in the presence of mAb specific for MHC class I, ICAM-1 and LFA-1 but not MHC class II. The relevance of these findings to T cell recognition of virus-infected cells in the central nervous system is discussed.