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Summary Disseminated gonococcal infection (DGI) is a rare but serious complication caused by the spread of Neisseria gonorrhoeae in the human host. Gonococci associated with DGI mainly express the outer membrane protein PorBIA that binds to the scavenger receptor expressed on endothelial cells (SREC-I) and mediates bacterial uptake. We recently demonstrated that this interaction relies on intact membrane rafts that acquire SREC-I upon attachment of gonococci and initiates the signalling cascade that finally leads to the uptake of gonococci in epithelial cells. In this study, we analysed the role of sphingomyelinases and their breakdown product ceramide. Gonococcal infection induced increased levels of ceramide that was enriched at bacterial attachment sites. Interestingly, neutral but not acid sphingomyelinase was mandatory for PorBIA-mediated invasion into host cells. Neutral sphingomyelinase was required to recruit the PI3 kinase to caveolin and thereby activates the PI3 kinase-dependent downstream signalling leading to bacterial uptake. Thus, this study elucidates the initial signalling processes of bacterial invasion during DGI and demonstrates a novel role for neutral sphingomyelinase in the course of bacterial infections.

Viral entry, compartmentalization and transmission depend on the formation of membrane lipid/protein microdomains concentrating receptors and signalosomes. Dendritic cells (DCs) are prime targets for measles virus (MV) infection, and this interaction promotes immune activation and generalized immunosuppression, yet also MV transport to secondary lymphatics where transmission to T cells occurs. In addition to MV trapping, DC-SIGN interaction can enhance MV uptake by activating cellular sphingomyelinases and, thereby, vertical surface transport of its entry receptor CD150. To exploit DCs as Trojan horses for transport, MV promotes DC maturation accompanied by mobilization, and restrictions of viral replication in these cells may support this process. MV-infected DCs are unable to support formation of functional immune synapses with conjugating T cells and signalling via viral glycoproteins or repulsive ligands (such as semaphorins) plays a key role in the induction of T-cell paralysis. In the absence of antigen recognition, MV transmission from infected DCs to T cells most likely involves formation of polyconjugates which concentrate viral structural proteins, viral receptors and with components enhancing either viral uptake or conjugate stability. Because DCs barely support production of infectious MV particles, these organized interfaces are likely to represent virological synapses essential for MV transmission.

Summary Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. We found that MV-infected DCs only subtly differed from LPS-matured with regard to integrin activation, acquisition of a migratory phenotype and motility. Similarly, the organization of MV-DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering and MHC class II surface recruitment. The majority of MV-DC/T cell conjugates was, however, unstable and only promoted abortive T cell activation. Thus, MV-infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilization and thereby act as effectors of T cell inhibition.

Summary Dendritic cells (DCs) act not only as sentinels for detection of, but also as target cells for viruses, and this can be important for viral transport and spread. All subsets of DCs are equipped with a battery of receptors recognizing virus-associated molecular signatures, and recognition of those launches a maturation programme that results in substantial alterations of morphology, motility and the DCs' interactive properties with the extracellular matrix and scanning T cells. In addition to being sensed, viruses are internalized into DCs and, for the major proportion, processed into peptides that are subsequently presented by major histocompatibility complex (MHC) molecules. Transmission of virus to T cells can occur after completion of their replication cycle if the intracellular milieu of the DC permits that. Alternatively, viruses can remain protected from degradation following entrapment by pattern recognition receptors in intracellular compartments, also referred to as virosomes, which translocate towards the DC/T cell interface. Most likely, transfer of virus to T cells occurs in these junctions, referred to as infectious synapses. In addition to promoting DC maturation, many viruses are able to downmodulate DC development and functions in order to evade immune recognition or to induce a generalized immunosuppression.

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in cis . Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.

T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca2+-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression., Author Summary Though the ability of measles virus (MV) to impair T cell activation has long been known, it is mechanistically not well understood. We have shown earlier that MV can contact dependently trigger activation of sphingomyelinases which is known to affect compartmentalization of membrane lipids and proteins. Because these are particularly important in the activity of the immune synapse (IS), we investigated whether MV-induced sphingomyelinase activity would interfere at that level with T cell activation. Our study for the first time revealed that the neutral sphingomyelinase 2 (NSM2) is transiently activated in primary T cells by co-stimulation through CD3 and CD28, and that this does occur to dampen early T cell responses. The virus appears to exploit this inhibitory activity of the enzyme to suppress T cell activation by promoting an enhanced and prolonged NSM2 activation. These findings do not only assign a hitherto novel role of the NSM2 in regulating T cell responses, but also reveal a novel strategy for viral T cell suppression.

By a contact-dependent surface interaction, the measles virus (MV) glycoprotein complex induces a pronounced inhibition of T-cell proliferation. We now show that MV directly interacts with glycosphingolipid-enriched membrane microdomains on human primary T cells and alters recruitment and segregation of membrane proximal signaling components. Contact-dependent interference with T-cell receptor-stimulated tyrosine phosphorylation and Ca mobilization is a late event seen 24 h after MV treatment. In contrast, stimulated recruitment of pleckstrin homology domain-containing proteins such as Akt and Vav is inhibited early after MV contact, as is segregation of the activated Akt kinase from rafts. Tyrosine phosphorylation of the regulatory subunit of the phosphatidylinositol 3-kinase (PI3K), p85, is apparently normal then, yet this protein fails to partition to the lipid raft fraction, and this is associated with stable expression of its negative regulator Cbl-b. Thus, by interaction with lipid rafts, MV contact initially targets recruitment of PI3K by preventing stimulated Cbl-b degradation and activation of PI3K-dependent signaling components.

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.

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.

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.

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the β-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.

The study of measles pathogenesis and the testing of improved vaccine candidates is hampered by the lack of a small animal model which is susceptible to infection by the intranasal route. With the identification of CD46 as a measles virus (MV) receptor, it was feasible to generate transgenic rats to overcome this problem. Although there was widespread expression of CD46 in the transgenic Sprague-Dawley rats, no measles-like disease could be induced after various routes of infection. The expressed transgenic protein was functionally intact since it mediated MV fusion and was downregulated by contact with MV hemagglutinin. In vitro studies revealed that CD46-expressing rat fibroblasts take up MV but do not allow viral replication, which explains the nonpermissiveness of the transgenic rats for in vivo infection.

Recently, it has been observed that the infection of human target cells with certain measles virus (MV) strains leads to the downregulation of the major MV receptor CD46. Here we report that CD46 downregulation can be rapidly induced in uninfected cells after surface contact with MV particles or MV-infected cells. Receptor modulation is detectable after 30 min of cocultivation of uninfected cells with MV-infected cells and is complete after 2 to 4 h, a time after which newly synthesized MV hemagglutinin (MV-H) cannot be detected in freshly infected target cells. This contact-mediated receptor modulation is also induced by recombinant MV-H expressed by vaccinia virus and is inhibitable with antibodies against CD46 and MV-H. By titrating the effect with MV Edmonston strain-infected cells, a significant contact-mediated CD46 modulation was detectable up to a ratio of 1 infected to 64 uninfected cells. As a result of CD46 downregulation, an increased susceptibility of uninfected cells for complement-mediated lysis was observed. This phenomenon, however, is MV strain dependent, as observed for the downregulation of CD46 after MV infection. These data suggest that in acute measles or following measles vaccination, uninfected cells might also be destroyed by complement after contacting an MV-infected cell.

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.

L'infection aigue par le virus de la rougeole et le deficit immunitaire transitoire qui l'accompagne sont encore a l'heure actuelle responsables de plus d'un million de deces chaque annee. L'atteinte aigue peut entrainer des complications touchant le systeme nerveux central. Nous resumons ici les aspects virologiques et immunologiques des infections aigues et persistantes par le virus de la rougeole. Plusieurs mecanismes contribuant a la persistance du virus dans le cerveau sont envisages. Les decouvertes recentes, telle l'analyse du recepteur du virus de la rougeole et la modulation de ce recepteur apres l'infection, auront des repercussions importantes sur la mise au point des prochains vaccins.

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).

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro . Infection of human CD34 + HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34 + CD150 − and CD34 + CD150 + (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150 + or CD150 − HS/PCs in vitro , it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.

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.

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics., Author Summary The ability of measles virus (MV) to impair T cell–dependent immune responses noted more than 100 years ago continues to be central to the severe generalised immunosuppression by this virus. Much has been learned about receptors and mechanisms, which determine the predilection of MV for hematopoetic cells. In contrast, little is known on a molecular level how MV interferes with processes relaying extracellular signals to T cells which translate into reorganisation of their cytoskeleton as required for their migration and cell–cell communication. Our study now shows that MV activates sphingomyelinases and ceramide accumulation in T cell membranes and this severely impairs integrity and stimulated reorganisation of their actin cytoskeleton, morphologically resulting in collapse of actin based protrusions, and functionally in impaired motility. During these studies, we appreciated, however, that cues other than MV eliciting ceramide accumulation in general also caused T cell paralysis. This indicates that ceramide accumulation and its consequences are not only a novel concept for MV-induced T cell silencing, but rather reflects a general strategy which may apply to extracellular ligands including pathogens able to promote plasma membrane ceramide accumulation, thereby preventing T cell activation.

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.

In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.

Summary Interference of measles virus (MV) with phosphatidyl-inositol-3-kinase (PI3K) activation in response to T cell receptor ligation was identified as important for the induction of T cell paralysis. We now show that MV exposure of unstimulated T cells induces expression of SIP110, an isoform of the lipid phosphatase SHIP145, which is translated from an intron-derived sequences containing mRNA. We found that MV contact can regulate stimulated exon inclusion into pre-mRNAs by targeting PI3K or MAPK-dependent nuclear translocation and activation of splicing regulatory serine–arginine rich (SR) and Sam68 proteins. Induction of SIP110 in resting T cells relied on MV-dependent interference with basal activity of the PI3K. SIP110 was cloned from MV-exposed T cells, and, when transiently expressed in primary or Jurkat T cells, localized into membrane clusters independently of T cell activation. Confirming that SIP110 is a catalytically active lipid phosphatase, its transgenic expression abolished basal and impaired PMA/ionomycin-stimulated phosphorylation of the Akt kinase which is important for T cell proliferation. Thus MV causes induction of SIP110 expression, which constitutively depletes the cellular phosphoinositol-3,4,5-phosphate pool suggesting that thereby the threshold for activation signals necessary for the induction of T cell proliferation is raised.

In contrast to studies with genetically modified viruses, RNA interference allows the analysis of virus infections with identical viruses and posttranscriptional ablation of individual gene functions. Using RNase III-generated multiple short interfering RNAs (siRNAs) against the six measles virus genes, we found efficient downregulation of viral gene expression in general with siRNAs against the nucleocapsid (N), phosphoprotein (P), and polymerase (L) mRNAs, the translation products of which form the ribonucleoprotein (RNP) complex. Silencing of the RNP mRNAs was highly efficient in reducing viral messenger and genomic RNAs. siRNAs against the mRNAs for the hemagglutinin (H) and fusion (F) proteins reduced the extent of cell-cell fusion. Interestingly, siRNA-mediated knockdown of the matrix (M) protein not only enhanced cell-cell fusion but also increased the levels of both mRNAs and genomic RNA by a factor of 2 to 2.5 so that the genome-to-mRNA ratio was constant. These findings indicate that M acts as a negative regulator of viral polymerase activity, affecting mRNA transcription and genome replication to the same extent.

In the course of acute measles, an efficient virus-specific immune response is generated which mediates viral clearance from the host and confers protection against reinfection. Paradoxically, a general immunosuppression is also induced favoring secondary infections, which are the major reason for the annual high morbidity and mortality rates associated with measles. The magnitude and duration of immune activation and immune suppression differ between natural or experimental infection and vaccination (20, 60). Studies addressing measles virus (MV)-induced immune suppression mainly have focused on alterations of T-cell functions and viability as a consequence of direct MV infection or contact-mediated signaling (53). In vitro observations suggest that MV infection also enhances apoptosis of monocytes and dendritic cells (DC) and affects their antigen-presenting capacity and cytokine release (31, 53). MV interaction with DC and monocytes is, however, also associated with their maturation or activation, respectively, and thus is important for induction of virus-specific immune responses (32, 39, 45, 54, 56). Strains expressing an MV wild-type-derived hemagglutinin (H) protein reveal a particular tropism for DC and are more efficient in inducing both DC maturation and immunosuppression (32, 48, 54). The mechanisms by which MV leads to these functional alterations are largely unknown. Downregulation of interleukin-12 (IL-12) production in monocytes was linked to MV- or antibody-mediated cross-linking of CD46, the receptor for certain MV strains (29). Lymphotropic MV wild-type strains and clinical isolates, with few known exceptions (43), fail to interact with CD46 but require CD150 for cell entry (15, 26, 49, 59). This molecule is absent from unstimulated monocytes and immature DC (33, 45, 48), and it is thus unknown how infection of these cells by CD150-dependent MV strains occurs. Mammalian Toll-like receptors (TLRs) were implicated in the innate immune recognition of a variety of bacterial pathogens and bacterial products (2). Ten TLR family members were discovered, and several of these appear to play important roles in the activation of cells by various bacterial products. TLR2 is responsible for recognition of gram-positive bacteria (57, 65), bacterial lipoproteins (12, 42), and lipoteichoic acid (38, 55). TLR4 appears to be the main receptor for lipopolysaccharide (LPS) lipid A from gram-negative bacteria (41), TLR6 might be a coreceptor for TLR2 in recognizing certain bacterial structures (50, 58), and TLR9 and TLR3 mediate responses to CpG bacterial DNA and double-stranded RNA (dsRNA), respectively (3, 24). Hence, these receptors are able to discriminate between different bacteria and bacterial structures and thereby direct a proper immune response to a specific pathogen. Intracellular domains of the TLRs share motifs with the protein families of the IL-1 receptors, and a common intracellular pathway leading to activation of NF-κB and mitogen-activated protein kinases involves MyD88, IRAK, and TRAF6 (2). However, other signaling pathways upstream of NF-κB have been described which also include utilization of the phosphatidylinositol-3/Akt-kinase pathway by TLR2 (4). It has recently been demonstrated that mammalian TLR signaling can also be regulated by viral gene products. Vaccinia virus encodes gene products that interfere with proximal TLR signaling in the cytoplasm (11), and the fusion protein of respiratory syncytial virus (RSV) was found to activate cells via TLR4 and CD14 (35). Using CHO cells stably overexpressing TLR2 or TLR4, we found that MV wild-type strains specifically activated cells via TLR2, and this was dependent on the expression of the H protein of the MV wild-type strain, WTF. The failure of attenuated MV strains to induce TLR2 activation correlated with a single amino acid exchange at position 481 which is, in turn, essential for the usage of CD46 as receptor by these strains. Importantly, MV expressing the wild-type H protein induced activation of TLR-responsive genes such as IL-1α/β, IL-6, and IL-12 p40 in monocytes and also the expression of CD150, the receptor for all MV strains. Activation of TLR signaling by wild-type MV H protein may thus essentially contribute to the immune activation during measles, and loss of the capability to activate TLR2 may be considered as an attenuation marker.

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 F0protein by cellular proteases, such as furin, into the F1-F2subunits 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 F0proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F0proteins was not due to an impaired F0-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 F0protein.

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.

Nonstructural proteins encoded by measles virus (MV) include the V protein which is translated from an edited P mRNA. V protein is not associated with intracellular or released viral particles and has recently been found to be dispensable for MV propagation in cell culture (H. Schneider, K. Kaelin, and M. A. Billeter, Virology 227:314–322, 1997). Using recombinant MVs (strain Edmonston [ED]) genetically engineered to overexpress V protein (ED-V+) or to be deficient for V protein (ED-V−), we found that in the absence of V both MV-specific proteins and RNAs accumulated to levels higher than those in the parental MV molecular clone (ED-tag), whereas MV-specific gene expression was strongly attenuated in human U-87 glioblastomas cells after infection with ED-V+. The titers of virus released from these cells 48 h after infection with either V mutant virus were lower than those from cells infected with ED-tag. Similarly, significantly reduced titers of infectious virus were reisolated from lung tissue of cotton rats ( Sigmodon hispidus ) after intranasal infection with both editing mutants compared to titers isolated from ED-tag-infected animals. In cell culture, expression of V protein led to a redistribution of MV N protein in doubly transfected Cos-7 cells, indicating that these proteins form heterologous complexes. This interaction was further confirmed by using a two-hybrid approach with both proteins expressed as Gal4 or VP16 fusion products. Moreover, V protein efficiently competed complexes formed between MV N and P proteins. These findings indicate that V protein acts to balance accumulation of viral gene products in cell culture, and this may be dependent on its interaction with MV N protein. Furthermore, expression of V protein may contribute to viral pathogenicity in vivo.

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.

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.

Transmission of measles virus (MV) to T cells by its early CD150 + target cells is considered to be crucial for viral dissemination within the hematopoietic compartment. Using cocultures involving monocyte-derived dendritic cells (DCs) and T cells, we now show that T cells acquire MV most efficiently from cis -infected DCs rather than DCs having trapped MV ( trans -infection). Transmission involves interactions of the viral glycoprotein H with its receptor CD150 and is therefore more efficient to preactivated T cells. In addition to rare association with actin-rich filopodial structures, the formation of contact interfaces consistent with that of virological synapses (VS) was observed where viral proteins accumulated and CD150 was redistributed in an actin-dependent manner. In addition to these molecules, activated LFA-1, DC-SIGN, CD81, and phosphorylated ezrin-radixin-moesin proteins, which also mark the HIV VS, redistributed toward the MV VS. Most interestingly, moesin and substance P receptor, both implicated earlier in assisting MV entry or cell-to-cell transmission, also partitioned to the transmission structure. Altogether, the MV VS shares important similarities to the HIV VS in concentrating cellular components potentially regulating actin dynamics, conjugate stability, and membrane fusion as required for efficient entry of MV into target T cells.

The alpha/beta (type I) interferon-inducible human MxA protein confers resistance to vesicular stomatitis virus (VSV) and influenza A virus in MxA-transfected mouse 3T3 cells (3T3/MxA). We investigated the inhibitory effects of the MxA protein on measles virus (MV) and VSV in the human monocytic cell line U937. In transfected U937 clones which constitutively express MxA (U937/MxA), the release of infectious MV and VSV was reduced approximately 100-fold in comparison with control titers. Transcription of VSV was inhibited similar to that observed for 3T3/MxA cells, whereas no difference was detected for MV in the rates of transcription or the levels of MV-specific mRNAs. In contrast, analysis of MV protein expression by immunofluorescence and immunoprecipitation revealed a significant reduction in the synthesis of MV glycoproteins F and H in U937/MxA cells. These data demonstrate a virus-specific effect of MxA which may, in the case of MV, contribute to the establishment of a persistent infection in human monocytic cells.

Application of neutralizing anti-hemagglutinin antibodies to mouse neuroblastoma cells (NS20Y/MS) persistently infected with measles virus (MV) leads to a significant reduction of viral structural proteins within 6 days. While the transcriptional gradient for MV-specific mRNAs remained unaffected upon antibody treatment, the total amount of MV-specific transcripts dropped by 80% after 24 h. The expression of genomic RNA was affected similarly, with slightly slower time kinetics. Both transcription and expression of the viral structural proteins could be completely reactivated when viral antibodies were removed from the tissue culture. The same findings could be obtained in rat glioma cells persistently infected with subacute sclerosing panencephalitis virus (C6/SSPE) but not in cells of nonneural origin. The data indicate that antibody-induced antigenic modulation affects the early stages of viral transcription within a few hours after the addition of antibodies and leads to an almost complete repression of viral gene expression in cells of neural origin.