ViralORFeome: an integrated database to generate a versatile collection of viral ORFs.

Large collections of protein-encoding open reading frames (ORFs) established in a versatile recombination-based cloning system have been instrumental to study protein functions in high-throughput assays. Such 'ORFeome' resources have been developed for several organisms but in virology, plasmid collections covering a significant fraction of the virosphere are still needed. In this perspective, we present ViralORFeome 1.0 (http://www.viralorfeome.com), an open-access database and management system that provides an integrated set of bioinformatic tools to clone viral ORFs in the Gateway(R) system. ViralORFeome provides a convenient interface to navigate through virus genome sequences, to design ORF-specific cloning primers, to validate the sequence of generated constructs and to browse established collections of virus ORFs. Most importantly, ViralORFeome has been designed to manage all possible variants or mutants of a given ORF so that the cloning procedure can be applied to any emerging virus strain. A subset of plasmid constructs generated with ViralORFeome platform has been tested with success for heterologous protein expression in different expression systems at proteome scale. ViralORFeome should provide our community with a framework to establish a large collection of virus ORF clones, an instrumental resource to determine functions, activities and binding partners of viral proteins.

System-level comparison of protein-protein interactions between viruses and the human type I interferon system network.

Innate immunity has evolved complex molecular pathways to protect organisms from viral infections. One pivotal line of cellular defense is the induction of the antiviral effect of interferon. To circumvent this primary response and achieve their own replication, viruses have developed complex molecular strategies. Here, we provide a systems-level study of the human type I interferon system subversion by the viral proteome, by reconstructing the underlying protein-protein interaction network. At this network level, viruses establish a massive and a gradual attack, from receptors to transcription factors, by interacting preferentially with highly connected and central proteins as well as interferon-induced proteins. We also demonstrate that viruses significantly target 22% of the proteins directly interacting with the type I interferon system network, suggesting the relevance of our network-based method to identify new candidates involved in the regulation of the antiviral response. Finally, based on the comparative analysis of interactome profiles across four viral families, we provide evidence of common and differential targeting strategies.

Serological and immunochemical analysis of the products of a single HLA DR-alpha and DR-beta chain gene expressed in a mouse cell line after DNA-mediated cotransformation reveals that the beta chain carries a known supertypic specificity.

Using a mouse cell line transformed with and expressing a single HLA DR-alpha and DR-beta chain gene, we present evidence that the product of the DR-beta chain gene carries a supertypic determinant, BR3, previously defined by serology. The amino acid sequence of this beta chain gene is determined from the DNA sequence. Another DR-associated supertypic specificity defined by monoclonal antibody MCS7 was not encoded by this DR-beta chain gene. This provides formal proof that a supertypic specificity can be associated with a product of a distinct DR-beta locus. We propose that haplotypes sharing such specificities are evolutionarily related.

Efficient major histocompatibility complex class II-restricted presentation of measles virus relies on hemagglutinin-mediated targeting to its cellular receptor human CD46 expressed by murine B cells.

Measles virus after binding to its cell surface human CD46 receptor fuses with the plasma membrane. This fusion results in envelope hemagglutinin (H) and fusion glycoprotein (F) incorporated into the plasma membrane and injection of the nucleocapsid made of nucleoprotein (NP) into the cytosol. The influence of targeting measles virus (MV) to CD46 in the processing and presentation of MV H and NP to antigen specific MHC class II I-E(d)- and I-A(d)-restricted T cell hybridomas was explored using murine M12-CD46 B cell transfectants. Parent M12 cells, which lack any MV receptor, were unable to present any of these two viral proteins when incubated with MV particles. Incubating M12.CD46 cells with 200 ng and 10 micrograms of MV could strongly stimulate H-specific and NP-specific T cells, respectively. Neosynthesis of MV proteins was not necessary since the efficiency of antigen presentation was similar when using ultraviolet-inactivated MV. Similar enhancing effects (more than 1,000-fold) on antigen presentation were also observed when using purified native H soluble or incorporated into liposomes whereas denaturating H glycoprotein resulted in a poor efficiency in T cell stimulation, M12.CD46 being no more potent than the parental M12 counterpart. MV H and NP presentation efficiency did not depend on MV fusion with plasma membrane as revealed by the lack of effect of specific fusion inhibitors. Both MV H and NP presentations were sensitive to chloroquine inhibition indicating that antigens from CD46-mediated captured MV were likely processed in the endosome/lysosome compartment. Altogether these data indicate that (a) MV targeting via CD46 has a strong effect on the efficiency of antigen presentation by MHC class II, (b) the effect is mediated by the binding of H to CD46, and (c) though MV does fuse with plasma membrane, endocytosis, and processing of virus particles are also occurring. Since, in humans, CD46 is expressed in almost every tissue including professional antigen-presenting cells, such a targeting is likely to play a crucial role in the CD4+ T cell-mediated primary immune response against the pathogen in vivo.

Measles virus nucleocapsid protein binds to FcgammaRII and inhibits human B cell antibody production.

Despite the development of an efficient specific immune response during measles virus (MV) infection, an immunosuppression occurs contributing to secondary infections. To study the role of nucleocapsid protein (NP) in MV-induced immunosuppression, we produced recombinant MV NP. Purified recombinant NP exhibited biochemical, antigenic, and tridimensional structure similar to viral NP. By flow cytometry, we showed that viral or recombinant NP bound to human and murine B lymphocytes, but not to T lymphocytes. This binding was specific, independent of MHC class II expression, and dependent of the B lymphocyte activation state. The murine IIA1. 6 B cell line, deficient in the Fc receptor for IgG (FcgammaRII) expression, did not bind NP efficiently. Transfected IIA1.6 cells expressing either murine FcgammaRIIb1 or b2, or human FcgammaRIIa, b1*, or b2 isoforms efficiently bound NP. Furthermore, this binding was inhibited up to 90% by monoclonal antibodies 2.4G2 or KB61 specific for murine and human FcgammaRII, respectively. Finally, the in vitro Ig synthesis of CD40- or Ig-activated human B lymphocytes in the presence of interleukin (IL)-2 and IL-10 was reduced by 50% in the presence of recombinant NP. These data demonstrate that MV NP binds to human and murine FcgammaRII and inhibits in vitro antibody production, and therefore suggests a role for NP in MV-induced immunosuppression.

Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells.

Secondary infections due to a marked immunosuppression have long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. The mechanisms underlying the inhibition of cell-mediated immunity are not clearly understood but dysfunctions of monocytes as antigen-presenting cells (APC) are implicated. In this report, we demonstrate that measles virus (MV) replicates weakly in the resting dendritic cells (DC) as in lipopolysaccharide-activated monocytes, but intensively in CD40-activated DC. The interaction of MV-infected DC with T cells not only induces syncytia formation where MV undergoes massive replication, but also leads to an impairment of DC and T cell function and cell death. CD40-activated DC decrease their capacity to produce interleukin (IL) 12, and T cells are unable to proliferate in response to MV-infected DC stimulation. A massive apoptosis of both DC and T cells is observed in the MV pulsed DC-T cell cocultures. This study suggests that DC represent a major target of MV. The enhanced MV replication during DC-T cell interaction, leading to an IL-12 production decrease and the deletion of DC and T cells, may be the essential mechanism of immunosuppression induced by MV.

Hepatitis C virus infection protein network.

A proteome-wide mapping of interactions between hepatitis C virus (HCV) and human proteins was performed to provide a comprehensive view of the cellular infection. A total of 314 protein-protein interactions between HCV and human proteins was identified by yeast two-hybrid and 170 by literature mining. Integration of this data set into a reconstructed human interactome showed that cellular proteins interacting with HCV are enriched in highly central and interconnected proteins. A global analysis on the basis of functional annotation highlighted the enrichment of cellular pathways targeted by HCV. A network of proteins associated with frequent clinical disorders of chronically infected patients was constructed by connecting the insulin, Jak/STAT and TGFbeta pathways with cellular proteins targeted by HCV. CORE protein appeared as a major perturbator of this network. Focal adhesion was identified as a new function affected by HCV, mainly by NS3 and NS5A proteins.

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 and dendritic cell functions: how specific response cohabits with immunosuppression.

Measles virus (MV) infection induces both an efficient MV-specific immune response and a transient but profound immunosuppression characterised by a panlymphopenia that occasionally results in opportunistic infections responsible for a high rate of mortality in children. On the basis of in vitro studies, the putative roles of dendritic cells (DCs) in MV infection are discussed. (1) DCs could participate in anti-MV innate immunity because MV turns on TNF-related apoptosis-inducing ligand (TRAIL)-mediated DC cytotoxicity. (2) Cross-priming by non-infected DCs might be the route of MV adaptive immune response. (3) After CD40-ligand activation in secondary lymphoid organs, MV-infected DCs could initiate the formation of Warthin-Finkeldey multinucleated giant cells, replicating MV and responsible for in vivo spreading of MV. (4) We review how integrated viral attack of the host immune system also targets DCs: Progress in understanding the immunobiology of MV-infected DCs that could account for MV-induced immunosuppression observed in vivo is presented and their potential role in lymphopenia is underlined. In conclusion, future research directions are proposed.

The MHC class II-associated invariant chain: a molecule with multiple roles in MHC class II biosynthesis and antigen presentation to CD4+ T cells.

The MHC class II-associated invariant chain (Ii) plays a central role in the biological function of MHC class II molecules. Ii is a type II membrane glycoprotein that is synthesized as different isoforms that include a major 31 kDa isoform (p31/p33) and a minor 41 kDa isoform (p41) in both, mice and humans. All isoforms share several common regions acting at different steps in the process that lead to functional class II molecule/peptide complexes. In the ER, two C-terminal extracytoplasmic regions of Ii are required for class II assembly: the 153-183 region is involved in the formation of Ii trimers and the 80-104 region mediates binding with class II molecules giving rise to nonamers. Ii association with class II molecules prevents both aggregation of class II dimers and binding with endogenous ER-derived peptides. In addition, two motifs in the cytosolic N-terminal region of Ii direct class II nonamers toward specialized endosomal compartments where peptide loading occurs. In these compartments, Ii undergoes proteolytic degradation leaving only CLIP (residues 80-104) associated with Class II. CLIP modulates loading of class II molecules in endosomes and is removed from the MHC class II groove by monomorphic MHC class II molecules, H2-M or HLA DM, in mouse and human, respectively. The roles of Ii in antigen presentation to MHC class II-restricted T cells and in CD4+ T cell development are discussed in this review.

Quantification of measles virus by a virus receptor-dependent and haemagglutinin-specific T cell stimulation assay.

The human measles virus receptor CD46 plays a major role in the uptake of measles virus (MV) for antigen presentation by major histocompatibility complex class II molecules to T cells. On this basis, a new bioassay has been set up to quantify measles virus in a cell free tissue culture supernatant. A stable mouse B cell transfectant expressing CD46 was used as the antigen presenting cell for presentation of measles virus to a haemagglutinin-specific and class II-restricted mouse T cell hybridoma. The measles virus haemagglutinin was quantified by its ability to stimulate IL-2 secretion by the T cells. A good correlation was found between the amount of haemagglutinin measured in supernatants from infected cells using the CD46-dependent T cell stimulation assay and the number of infectious viral particles as determined in a plaque assay. When MV was purified on a discontinuous sucrose gradient, most of the infectious virus and the haemagglutinin antigen were recovered in the same fraction. These data indicate that the CD46-dependent haemagglutinin-specific T cell assay could be used to measure the production of measles virus in the supernatant of infected cells. The assay required only 48 h, was sensitive, highly specific, and did not rely on the replication of the virus. This new bioassay would be applicable for the detection of any other virus provided that antigen presenting cells expressing the corresponding virus receptor and virus envelope glycoprotein-specific T cells are available. Moreover, it would be an interesting tool to monitor the receptor binding properties of attenuated vaccine virus and envelope glycoprotein subunit vaccines.

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex.

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.

Tumor-promoting phorbol diesters inhibit in vitro antibody synthesis.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate prevents the synthesis of antibodies directed against sheep red blood cells in an in vitro system. The inhibitory effect of 3 phorbol diesters on this immune response was positively correlated with their tumor-promoting activity. The effect did not appear to be mediated through the inhibition of cell proliferation. Results suggest that the tumor promoter may alter the differentiation of the precursor cells to antibody-producing cells.

Measle virus-infected dendritic cells develop immunosuppressive and cytotoxic activities.

Measle virus (MV) infection induces a transient but profound immunosuppression characterized by a panlymphopenia which occasionally results in opportunistic infections responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DC) in the respiratory mucosa or in the secondary lymphoid organs. After a brief presentation of DCs, we review progress in understanding the immunobiology of MV-infected DCs that could account for MV-induced immunosuppression. In addition, we develop the newly described TRAIL-mediated cytotoxic function of DCs that is turned on by MV infection, but also by interferons or double-stranded RNA (poly (I:C)). Finally, we propose a model where the measles-associated lymphopenia could be mediated by TRAIL and the measles-induced immunosuppression could be transiently prolonged by Fas-mediated destruction of DCs.

sst5 somatostatin receptor mRNA induction by mitogenic activation of human T-lymphocytes.

SRIF has neuro-immunomodulatory actions on immune cells, including T-lymphocytes. Molecular mechanisms involved in these actions were studied by RT-PCR analysis of SRIF receptor expression in resting and initogen-activated human T-lymphocytes. Our results point to the mitogen-associated induction of sst5 receptor subtype. Conversely, sst3 receptor appears constitutively expressed in both activity states. Assessment of biologic actions of SRIF14 in activated T-lymphocytes indicates that, in nanomolar concentration range, this peptide moderately inhibits mitogen-induced IL-2 secretion. Nevertheless, T-lymphocyte proliferation is not inhibited in the presence of SRIF14 but is even slightly increased. Altogether these data suggest a complex mechanism of SRIF neuro-immunomodulatory actions.

Critical residue combinations dictate peptide presentation by MHC class II molecules.

Peptides encompassing the core hen egg lysozyme HEL(52-61) peptide elongated or not and substituted or not with natural and unnatural amino acids were used to find a peptide motif for binding to the major histocompatibility complex (MHC) class II I-Ak. Using a T-cell recognition functional assay, nine out of 10 positions were found to be somehow involved in the I-Ak binding, and six out of 10 residues were involved in T-cell recognition. The deleterious effect of single substitutions could be rescued by changing peptide length and/or sequence. Thus, efficient binding to MHC class II molecules requires not only few anchoring residues correctly interspaced, but a complex, nonrandom combination of residues with appropriate orientation of the peptide backbone and some crucial side chains.

Mode of entry of morbilliviruses.

The morbilliviruses have a restricted host range. This is probably dependent on the use of specific host cell receptors. In the present article, we have reviewed our approach to identify a host cell receptor for one of the morbilliviruses, measles virus and to elucidate the interaction between viral and cellular proteins during virus entry into the host cell.

Genome-wide expression analyses establish dendritic cells as a new osteoclast precursor able to generate bone-resorbing cells more efficiently than monocytes.

Dendritic cells (DCs), mononuclear cells that initiate immune responses, and osteoclasts (OCs), multinucleated bone-resorbing cells, are hematopoietic cells derived from monocytic precursor cells. Using in vitro generated dendritic cells, we previously showed that human and murine DCs could transdifferentiate into resorbing osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). In this study we globally compared by transcriptomic profiling this new osteoclast differentiation pathway from DCs with the canonical differentiation pathway from monocytes. DNA chip data revealed that starting from two very distinct cell types, treatment with M-CSF and RANKL generated two highly similar types of osteoclast. In particular, DC-derived osteoclasts expressed all the characteristic marker genes of monocyte-derived osteoclasts. Two major molecular events could be observed during osteoclastogenesis: downregulation of a large set of monocyte or DC specific markers, together with upregulation of characteristic osteoclast marker genes. Most interestingly, our transcriptomic data showed a closer molecular profile between DCs and OCs than between monocytes and OCs. Our data establish DCs as a new osteoclast precursor able to generate OCs more efficiently than monocytes.