Measles virus interaction with host cells and impact on innate immunity.

Because viruses are obligate parasites, numerous partnerships between measles virus and cellular molecules can be expected. At the entry level, measles virus uses at least two cellular receptors, CD150 and a yet to be identified epithelial receptor to which the virus H protein binds. This dual receptor strategy illuminates the natural infection and inter-human propagation of this lymphotropic virus. The attenuated vaccine strains use CD46 as an additional receptor, which results in a tropism alteration. Surprisingly, the intracellular viral and cellular protein partnership leading to optimal virus life cycle remains mostly a black box, while the interactions between viral proteins that sustain the RNA-dependant RNA polymerase activity (i.e., transcription and replication), the particle assembly and the polarised virus budding are documented. Hsp72 is the only cellular protein that is known to regulate the virus transcription and replication through its interaction with the viral N protein. The viral P protein is phosphorylated by the casein kinase II with undetermined functional consequences. The cellular partnership that controls the intracellular trafficking of viral components, the assembly and/or the budding of measles virus, remains unknown. The virus to cell innate immunity war is better documented. The 5' triphosphate-ended virus leader transcript is recognised by RIG-I, a cellular helicase, and induces the interferon response. Measles virus V protein binds to the MDAS helicase and prevents the MDA5-mediated activation of interferon. By interacting with STAT1 and Jak1, the viral P and V proteins prevent the type I interferon receptor (IFNAR) signalling. The virus N protein interacts with eIF3-p40 to inhibit the translation of cellular mRNA. The H protein binds to TLR2, which then transduces an activation signal and CD150 expression in monocytes. The P protein activates the expression of the ubiquitin modifier A20, thus blocking the TLR4-mediated signalling. Few other partnerships between measles virus components and cellular proteins have been postulated or demonstrated, and they need further investigations to understand their physiopathological outcome.

[A cellular receptor to get in, another to get out]. / Un récepteur pour entrer, un second pour sortir.

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Cell-cell fusion induced by measles virus amplifies the type I interferon response.

Measles virus (MeV) infection is characterized by the formation of multinuclear giant cells (MGC). We report that beta interferon (IFN-beta) production is amplified in vitro by the formation of virus-induced MGC derived from human epithelial cells or mature conventional dendritic cells. Both fusion and IFN-beta response amplification were inhibited in a dose-dependent way by a fusion-inhibitory peptide after MeV infection of epithelial cells. This effect was observed at both low and high multiplicities of infection. While in the absence of virus replication, the cell-cell fusion mediated by MeV H/F glycoproteins did not activate any IFN-alpha/beta production, an amplified IFN-beta response was observed when H/F-induced MGC were infected with a nonfusogenic recombinant chimerical virus. Time lapse microscopy studies revealed that MeV-infected MGC from epithelial cells have a highly dynamic behavior and an unexpected long life span. Following cell-cell fusion, both of the RIG-I and IFN-beta gene deficiencies were trans complemented to induce IFN-beta production. Production of IFN-beta and IFN-alpha was also observed in MeV-infected immature dendritic cells (iDC) and mature dendritic cells (mDC). In contrast to iDC, MeV infection of mDC induced MGC, which produced enhanced amounts of IFN-alpha/beta. The amplification of IFN-beta production was associated with a sustained nuclear localization of IFN regulatory factor 3 (IRF-3) in MeV-induced MGC derived from both epithelial cells and mDC, while the IRF-7 up-regulation was poorly sensitive to the fusion process. Therefore, MeV-induced cell-cell fusion amplifies IFN-alpha/beta production in infected cells, and this indicates that MGC contribute to the antiviral immune response.

Measles virus protein interactions in yeast: new findings and caveats.

Complementary DNA clones of measles virus N, N (S228Q; L229D), Ncore (N1-400), Ntail (N401-525), P, PNT (P1-230), PCT (P231-507), L, MEL (L800-2183) and EL (L1300-2183) were fused in frame downstream of the Gal4 binding domain (BD) or activating domain (AD). All but BD-L, BD-MEL and BD-EL, were detected by western blot, with additional C- and/or N-terminal truncated products in the case of BD-N, and BD-P. BD-P and BD-PNT directly activated the reporter genes, indicating that the PNT domain displays transactivating properties. In yeast two-hybrid assays, PNT and PCT domains bind to Ncore and Ntail domains, respectively, indicating that N and P interact in a head to tail orientation via two independent binding sites. BD-N (S228Q; L229D) and AD-N displayed no or poor interaction with P proteins possibly because they may not be properly folded. L binding site on P lies within the PCT domain, and two PCT binding sites lie within the L1-799 and L800-1300 regions. Thus, N to P and P to L protein interactions in measles virus shared many features with other related Paramyxoviridae. From a human cDNA library, several candidate partners of N protein were identified which all reacted with BD-Ncore, and RNA was found to bridge the N protein with one partner.

Evidence for distinct complement regulatory and measles virus binding sites on CD46 SCR2.

Human CD46, or membrane cofactor protein, is a regulator of complement activation and is used as a cellular receptor by measles virus. Using a series of 13 single point mutants, the region of short consensus repeat (SCR) 2 domain involved in the regulation of complement activation was mapped to residues E84, N94, Y98, E102, E103, I104 and E108. Molecular modelling localized all residues, with the exception of E84, close to each other on the external lateral face of the molecule, away from the residues important for the binding of measles virus, which are localized on the top of the molecule. The E84 residues is localized in the SCR1-2 hinge and the deleterious effect of its substitution by an alanine residue could affect the relative orientation and / or tilt of SCR1 on SCR2. Taken together, the results suggest that the measles virus binding and cofactor activity of CD46 map to distinct areas on the SCR2 module.

Measles virus assembly within membrane rafts.

During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305-311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

CD40 signaling in human dendritic cells is initiated within membrane rafts.

Despite CD40's role in stimulating dendritic cells (DCs) for efficient specific T-cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte-derived DCs associate with sphingolipid- and cholesterol-rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft-associated Lyn Src family kinase, and possibly other raft-associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft-restricted recruitment of tumor necrosis factor (TNF) receptor-associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40's cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)-1alpha/beta and IL-1Ra mRNA production and contributes to p38-dependent IL-12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40-mediated pathways on cytokine production.

Octamerization enables soluble CD46 receptor to neutralize measles virus in vitro and in vivo.

A chimeric fusion protein encompassing the CD46 ectodomain linked to the C-terminal part of the C4b binding protein (C4bp) alpha chain (sCD46-C4bpalpha) was produced in eukaryotic cells. This protein, secreted as a disulfide-linked homo-octamer, was recognized by a panel of anti-CD46 antibodies with varying avidities. Unlike monomeric sCD46, the octameric sCD46-C4bpalpha protein was devoid of complement regulatory activity. However, sCD46-C4bpalpha was able to bind to the measles virus hemagglutinin protein expressed on murine cells with a higher avidity than soluble monomeric sCD46. Moreover, the octameric sCD46-C4bpalpha protein was significantly more efficient than monomeric sCD46 in inhibiting virus binding to CD46, in blocking virus induced cell-cell fusion, and in neutralizing measles virus in vitro. In addition, the octameric sCD46-C4bpalpha protein, but not the monomeric sCD46, fully protected CD46 transgenic mice against a lethal intracranial measles virus challenge.

CD46 (membrane cofactor protein) associates with multiple beta1 integrins and tetraspans.

The tetraspans associate with a large number of surface molecules, including a subset of beta1 integrins and, indirectly through CD19, with the complement receptor CD21. To further characterize the tetraspan complexes we have raised and selected monoclonal antibodies (mAb) for their ability to immunoprecipitate a molecule associated with CD9. A unique mAb was identified which recognizes the complement regulator CD46 (membrane cofactor protein). CD46 associated in part with several tetranspans and with all beta1 integrins that were tested (CD29/CD49a, CD29/CD49b, CD29/CD49c, CD29/CD49e, CD29/CD49f) but not with beta4 integrins. These data, together with cross-linking experiments showing the existence in living cells of CD46/integrin complexes, suggest that CD46 associates directly with beta1 integrins and indirectly with tetraspans. CD46 also acts as a receptor for measles virus; however, mAb to various integrins and tetraspans did not modify the virus fusion entry step.

Interaction of CD46 with measles virus: accessory role of CD46 short consensus repeat IV.

To define further the accessory role(s) of the CD46 (membrane cofactor protein) short consensus repeat (SCR) III and IV domains in the interaction of CD46 with measles virus (MV), chimeric proteins were generated by substituting domains from the structurally related protein decay accelerating factor (DAF, CD55): x3DAF (exchange of CD46 SCR III) and x4DAF (exchange of SCR IV). Transfected CHO cell lines that stably expressed these chimeric proteins were compared for MV binding and infection. Compared with wild-type CD46 (I-II-III-IV), a significant decrease in MV binding was observed with x4DAF. Despite this limited binding, these cells were still capable of supporting virus entry. In a quantitative fusion assay, no significant differences in fusion were observed as a result of the exchange of either CD46 SCR III or IV. However, the down-regulation of cell surface CD46 typically observed following MV infection was abolished with x4DAF, as was the redistribution of CD46 on the cell surface. Thus, CD46 SCR IV appears to be required for optimal virus binding and receptor down-regulation, although importantly, in spite of these functional limitations, x4DAF can still be used for MV entry.

Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly.

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.

Chimeric CD46/DAF molecules reveal a cryptic functional role for SCR1 of DAF in regulating complement activation.

Chimeric proteins using membrane cofactor (CD46) and decay accelerating factor (DAF or CD55) were generated to further investigate the functional domains involved in the regulation of human serum complement. Following activation of the classical pathway, the isolated substitution of CD46 SCR III (x3DAF) exhibited a modest regulatory activity comparable to that of CD46. The isolated substitution of CD46 SCR IV (x4DAF), and the combined CD46 SCR III+IV substitutions (x3/4DAF) were essentially as efficient as DAF. No regulation of C3b deposition was observed with the combined CD46 SCR I+II substitutions (x1/2DAF). When tested after activation of the alternative pathway, both the x3DAF and x3/4DAF chimeras failed to regulate C3b deposition, while the x4DAF chimera still displayed some activity. In contrast to that observed following classical pathway activation, the x1/2DAF chimera exhibited a similar efficiency to wild type CD46 and DAF in controlling C3b deposition. Using SCR specific antibodies, the regulatory activity of the x1/2DAF chimera against the alternative pathway was mapped to the first three distal SCR (i.e. DAF 1, DAF 2 and CD46 III). These data demonstrate that several combinations of SCR domains from two related complement regulators can result in functional molecules, and reveal a novel and cryptic functional role for DAF SCR1.

Conformational restriction of the Tyr53 side-chain in the decapeptide HE.

A series of conformationally restricted analogs of the hen egg lysozyme (HEL) decapeptide 52-61 in which the conformationally flexible Tyr53 residue was replaced by several more constrained tyrosine and phenylalanine analogs was prepared. Among these tyrosine and phenylalanine analogs were 1,2,3,4-tetrahydro-7-hydroxyisoquinoline-3-carboxylic acid (Htc), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 4-amino- 1,2,4,5-tetrahydro-8-hydroxy-2-benzazepine-3-one (Hba), 4-amino-1,2,4,5-tetrahydro-2-benzazepine-3-one (Aba), 2-amino-6-hydroxytetralin-2-carboxylic acid (Hat) and 2-amino-5-hydroxyindan-2-carboxylic acid (Hai) in which the rotations around Calpha-Cbeta and Cbeta-Cgamma were restricted because of cyclization of the side-chain to the backbone. Synthesis of Pht-Hba-Gly-OH using a modification of the Flynn and de Laszlo procedure is described. Analogs of beta-methyltyrosine (beta-MeTyr) in which the side-chains were biased to particular side-chain torsional angles because of substitution at the beta-hydrogens were also prepared. These analogs of HEL[52-61] peptide were tested for their ability to bind to the major histocompatibility complex class II I-Ak molecule and to be recognized in this context by two T-cell hybridomas, specific for the parent peptide HEL[52-61]. The data showed that the conformation and also the configuration of the Tyr53 residue influenced both the binding of the peptide to I-Ak and the recognition of the peptide/I-Ak complex by a T-cell receptor.

Inefficient measles virus budding in murine L.CD46 fibroblasts.

Infection of mouse L.CD46 fibroblasts with measles virus resulted in a poor virus yield, although no defects in the steps of virus binding, entry or fusion, were detected. Two days post-infection, the level of expression of the viral F protein was found to be similar on the surface of infected L.CD46 and HeLa cells using a virus multiplicity enabling an equal number of cells to be infected. After immunofluorescence labelling and confocal microscopy, L.CD46 cells also displayed a significant increase in the co-localisation of the N protein with the cell surface H and F proteins. Immunogold labelling and transmission electron microscopy demonstrated the accumulation of numerous nucleocapsids near the plasma membrane of L. CD46 cells with little virus budding, in contrast to infected HeLa cells which displayed fewer cortical nucleocapsids and more enveloped viral particles. Purified virus particles from infected L. CD46 contained a reduced amount of H, F and M protein. Altogether, these data indicate that, in L.CD46 cells, the late stage of measles virus assembly is defective. This cellular model will be helpful for the identification of cellular factors controlling measles virus maturation.

Infection of chicken embryonic fibroblasts by measles virus: adaptation at the virus entry level.

Measles virus (MV) has a tropism restricted to humans and primates and uses the human CD46 molecule as a cellular receptor. MV has been adapted to grow in chicken embryonic fibroblasts (CEF) and gave rise to an attenuated live vaccine. Hallé and Schwarz MV strains were compared in their ability to infect both simian Vero cells and CEF. Whereas both strains infected Vero cells, only the CEF-adapted Schwarz strain was able to efficiently infect CEF. Since the expression of the human MV receptor CD46 rendered the chicken embryonic cell line TCF more permissive to the infection by the Hallé MV strain, the MV entry into CEF appeared to be a limiting step in the absence of prior MV adaptation. CEF lacked reactivity with anti-CD46 antibodies but were found to express another protein allowing MV binding as an alternative receptor to CD46.

Control of C3b and C5b deposition by CD46 (membrane cofactor protein) after alternative but not classical complement activation.

C3b and C5b deposition following complement activation, and its regulation by CD46 were studied using xenogenic Chinese hamster ovary (CHO) cells as targets and cytofluorometry. Following activation of the alternative pathway, an initial low level of C3b deposition was observed on CHO cell surfaces after a lag time of approximately 4 min. This was followed by a secondary high level of C3b deposition with a slower rate. C3b deposition was maximal within 15 min. When CD46 was expressed (B2 isoform), the kinetics of C3b deposition were essentially unchanged, but the onset of the secondary high C3b deposition was fully prevented. C5b deposition was also observed on CHO but not on CHO.CD46 cells following activation of the alternative pathway. Activation of the classical pathway on CHO and CHO.CD46 cells, using factor B-depleted human serum and anti-CHO antibodies, resulted in almost identical single-peak C3b deposition profiles. Accordingly, no regulation of C5b deposition by CD46 was evident following activation of the classical pathway. These data indicate that CD46 prevents the C3b deposition amplification loop mediated by the alternative C3 convertase and, consequently, inhibits the formation of the alternative C5 convertase. But CD46 prevents neither the spontaneous tick-over C3b deposition leading to the formation of the alternative C3 convertase nor the formation of the functional classical C3 and C5 convertases.

Nonstructural C protein is required for efficient measles virus replication in human peripheral blood cells.

The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V- was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C- progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C--infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C- was as potent as MV tag or MV V- in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.

An accessory peptide binding site with allosteric effect on the formation of peptide-MHC-II complexes?

MHC-II molecules bind a single peptide in their groove. Here, the authors summarise evidence that a second peptide could bind transiently to MHC-II molecules outside the groove and have an allosteric effect on peptide-MHC-II complex formation. This effect could modulate, after the antigen processing, the selection of the peptide subset presented by MHC-II molecules to the helper CD4 T cells, which regulate the specific immune response.