Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2^−/−mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.

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.

As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DC-SIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DC-SIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses. Dendritic cells (DCs) bear receptors specialized on recognition of patterns specific to pathogens (such as carbohydrates), which can either promote functional activation of these cells (such as TLRs), which renders them capable of efficiently presenting antigens to T cells, or, as DC-SIGN, endocytic uptake as essential for loading MHC molecules. Viruses such as HIV and measles virus (MV) exploit DC-SIGN for both their uptake into DCs and modulation of TLR signaling, yet how this is mechanistically exerted is poorly understood. We now show that DC-SIGN activates sphingomyelinases (SMases) which convert their sphingomyelin substrate into ceramides, thereby catalysing the formation of membrane platforms able to recruit and concentrate receptors and associated signaling components. We found DC-SIGN-dependent SMase activation as essential for DC-SIGN and thereby modulation of TLR signaling, but also for enhancement of MV uptake. This is mediated by a fast, transient recruitment of its entry receptor, CD150, from an intracellular storage compartment to the cell surface where it co-clusters in ceramide enriched platforms with DC-SIGN. The ability to segregate viral receptors into (or exclude them from) membrane microdomains, which, based on their biophysical properties, facilitate membrane fusion, proposes DC-SIGN-mediated SMAse activation as a central regulator of pathogen uptake into DCs.

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. 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.

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.