Identification of new benzofuran derivatives as STING agonists with broad-spectrum antiviral activity.

The Stimulator of Interferon Genes (STING) is involved in cytosolic DNA sensing and type I Interferons (IFN-I) induction. Aiming to identify new STING agonists with antiviral activity and given the known biological activity of benzothiazole and benzimidazole derivatives, a series of benzofuran derivatives were tested for their ability to act as STING agonists, induce IFN-I and inhibit viral replication. Compounds were firstly evaluated in a gene reporter assay measuring luciferase activity driven by the human IFN-ß promoter in cells expressing exogenous STING (HEK293T). Seven of them were able to induce IFN-ß transcription while no induction of the IFN promoter was observed in the presence of a mutated and inactive STING, showing specific protein-ligand interaction. Docking studies were performed to predict their putative binding mode. The best hit compounds were then tested on human coronavirus 229E replication in BEAS-2B and MRC-5 cells and three derivatives showed EC50 values in the µM range. Such compounds were also tested on SARS-CoV-2 replication in BEAS-2B cells and in Calu-3 showing they can inhibit SARS-CoV-2 replication at nanomolar concentrations. To further confirm their IFN-dependent antiviral activity, compounds were tested to verify their effect on phospho-IRF3 nuclear localization, that was found to be induced by benzofuran derivatives, and SARS-CoV-2 replication in Vero E6 cells, lacking IFN production, founding them to be inactive. In conclusion, we identified benzofurans as STING-dependent immunostimulatory compounds and host-targeting inhibitors of coronaviruses representing a novel chemical scaffold for the development of broad-spectrum antivirals.

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.

An interdisciplinary approach to controlling chikungunya outbreaks on French islands in the south-west Indian ocean.

The outbreak of chikungunya that occurred on French Island territories in the southwest Indian Ocean in 2005 and 2006 caused severe morbidity and mortality. In the aftermath, French authorities set up a scientific task force including experts in epidemiology, public health, entomology, virology, immunology, sociology, animal health, community and hospital medicine. The mission of the task force was to conceive and propose research programs needed to increase understanding of the disease and epidemic and to help public health officials in improving epidemic response measures. The purpose of this article is to describe the findings of the task force at the end of its two-year existence and initial outcomes in the the areas studied. Discussion emphasizes topics requiring further study.

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.

Modulation of HLA class II antigen expression by transfection of sense and antisense DR alpha cDNA.

In the human there are three isotypic forms of MHC class II gene products (HLA-DR, -DQ, and -DP). The isotype-matched alpha-beta dimers are predominant but isotype-mismatched dimers can also be expressed (DR alpha-DQ beta). Here it is shown that the expression of the DR alpha-DQ beta dimer can be correlated to a high ratio of DR alpha/DR beta mRNA. The DR alpha chain expression was modulated by transfection of a sense and antisense DR alpha cDNA. Overexpression of DR alpha promoted the appearance of the DR alpha-DQ beta dimer. On the other hand, pre-existing DR alpha-DQ beta dimer expression was suppressed after antisense DR alpha cDNA transfection. Therefore, imbalanced expression of the alpha and beta chain from a given isotype could lead to the modification of HLA class II phenotype.

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.

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.

A quantitative dot immunobinding assay for human HLA class II antigens using nitrocellulose membrane filters.

A quantitative method for the evaluation of human HLA-DR antigen expression has been developed. Cell membrane proteins were solubilized in Nonidet P-40 or deoxycholic acid detergent and diluted in a Triton X-100 containing sample buffer. The samples were subsequently spotted on a nitrocellulose membrane filter and fixed by immersion in isopropyl alcohol-acetic acid solution. The membrane was saturated in a 5% BSA blocking buffer and sequentially incubated with specific monoclonal anti-HLA-DR antibody, and 125I-labelled protein A. Each spot was then assayed for radioactivity in a gamma scintillation counter. Immunoadsorbant purified HLA-DR antigen was used to standardize the method and a reference dosage curve was established with serial dilutions of the purified HLA-DR antigen. The method permitted the detection of HLA-DR antigens with reproducibility in the ng range, in cellular extracts, physiological and pathological fluids, and in fractions eluted from affinity columns.

Protection against experimental autoimmune encephalomyelitis by a proteasome modulator.

The capacity of interferon beta to alter the course of multiple sclerosis has promoted a new therapeutic concept, based upon the modulation of the immune response rather than its suppression. As the proteasome plays a crucial role in the control of the inflammatory process and immune cell survival, targeting the proteasome appears as a novel approach for the prevention and treatment of inflammatory autoimmune diseases. We have previously shown that ritonavir, an HIV-1 protease inhibitor used in AIDS therapy, can modulate the proteasome function by inhibiting the chymotrypsin-like activity and enhancing the trypsin-like activity. We have, therefore, explored its therapeutic potential on experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis, in Lewis rats and SJL mice. Daily administration of ritonavir during autoimmune antigen stimulation prevented clinical symptoms of EAE in a dose- and time-dependent manner. This protection was accompanied by an inhibition of the mononuclear cell infiltration into the central nervous system usually observed in EAE. Despite a complete absence of clinical symptoms during first EAE induction, ritonavir-treated animals became resistant to further induction of EAE, suggesting an immune mechanism of protection. These results suggest that proteasome modulation using ritonavir or analogues may be of interest for patients with multiple sclerosis.

Quantitation of HCV RNA using real-time PCR and fluorimetry.

Real-time PCR technology may provide an accurate and sensitive method to quantify hepatitis C virus (HCV) RNA. So far, studies have been carried out using the Taqman technology with the ABI Prism 7700 sequence detector. An alternative and simple real-time PCR assay is described with no probe requirement, based on the SYBR Green I dye and LightCycler fluorimeter. Amplicon synthesis was monitored continuously by SYBR Green I dye binding to double stranded DNA during PCR of the 5' HCV non-coding (NC) region. Specificity was verified by amplicon melting temperatures. An external standard curve was constructed with serial 10 fold dilutions of a modified synthetic HCV 5' NC RNA. A wide range linear relationship (up to 3.7x10(9) copies/ml) was observed between number of PCR cycle needed to detect a fluorescent signal and number of RNA copy. Intra- and inter-assay coefficients of variation were 0.7 to 2.1 and 3.7% respectively, indicating good reproducibility of the method. Thirty-three HCV positive sera of different genotypes were quantified by this method and gave similar but more sensitive results compared to the branched DNA (bDNA) technology.

Selective increased presentation of type II collagen by leupeptin.

Type II collagen (CII) is an arthritogenic self antigen in DBA/1 (H-2q) mice. To analyze the intracellular processing of this fibrillar protein in the context of I-Aq molecules, we have generated hybrid antigen-presenting cells (APC) by fusion of B lymphoma (A20 and M12) cells with CII-primed spleen cells from DBA/1 mice. Efficient presentation of CII by these APC to specific T cell hybridomas required prior cleavage of the antigen and intracellular handling of the peptides. Inhibition of protein transport by brefeldin A prevented the presentation of CII peptides to T cell hybridomas, indicating that the intracellular presentation of CII was dependent on neo-synthesis of I-Aq molecules. In contrast, exposure of hybrid B lymphomas to leupeptin, a protease inhibitor, induced a dose-dependent increase of CII-specific T cell response, while abrogating the I-Aq-restricted presentation of ovalbumin. The enhancing effect of leupeptin was also observed when immune B cells were used as APC. In contrast, leupeptin inhibited the presentation of CII peptides by macrophages or total spleen cells. Pulse-chase analysis of metabolically labeled hybrid APC and immunoprecipitation with antibodies specific for class II molecules or invariant (li) chain revealed that leupeptin did not affect the li chain processing or the formation of stable class II dimers. The stimulatory effect of leupeptin observed on CII presentation suggests that leupeptin protects CII epitopes by interfering with proteases involved in the intracellular degradation of CII.

Xenogeneic recognition of soluble and cell surface HLA class II antigens by proliferative murine T cells.

In order to characterize the murine anti-human xenogeneic mixed lymphocyte reactions (MLR), we studied T cell proliferative responses against various human lymphoid cells by immunization of mice either with cellular or purified HLA-DR antigens. Data presented here indicated that small amounts of soluble HLA-DR antigen were able to prime mice, and that the xenogeneic MLR depends on the expression of HLA class II antigens on the stimulating cells. Experiments using a mutant cell line clearly showed that HLA-DP molecules were also sufficient in eliciting a primary or a secondary xenogeneic MLR while no secondary proliferative response was obtained with cells expressing only HLA class I molecules. Using a large panel of human cells with various haplotypes, our results also showed that (a) nonpolymorphic determinants of HLA class II antigens trigger dominantly the murine T cells and (b) the xenogeneic response required I-E and L3T4 accessory molecules and was not inhibited with anti I-A and monomorphic anti-HLA class II antigen monoclonal antibodies. Altogether these results suggest that HLA class II antigens act as nominal antigens in triggering a murine anti-human proliferative response.

A novel HLA class II molecule (DR alpha-DQ beta) created by mismatched isotype pairing.

Human major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins composed of non-covalently associated alpha and beta chains. Only isotype-matched alpha-beta associations have been described in man; these can occur either by cis- or trans-complementation (HLA-DR, DQ, DP). Here evidence is provided for the existence of a new type of hybrid molecule (DR alpha-DQ beta) arising by mixed-isotype pairing in human B-cell lines. Class II isotype-mismatched heterodimers have been recently reported in the mouse after transfection of class II genes, and our data demonstrate that such interisotypic pairing can occur in untransfected cells. This crosspairing greatly enhances the repertoire of the class II antigens that regulate immune responses and leads us to reconsider the HLA-disease association.

Hybrid HLA-DC antigens provide molecular evidence for gene trans-complementation.

In the mouse there are hybrid determinants for the immune region (Ir) of the genome which contribute to the diversity of class II (Ia) antigens of the major histocompatibility complex (MHC) and provide a molecular basis for Ir gene complementation. In man, two prominent families of Ia molecule, HLA-DR and HLA-DC (or DS), have been identified which are respectively homologous to the murine I-E (E alpha, E beta) and I-A (A alpha, A beta) antigens. Whereas DR antigens consist of a constant alpha-chain and an extremely polymorphic beta-chain, both the alpha and beta subunits of DC antigens are structurally variable when different alleles are compared. The marked differences in the structure of the alpha- and beta-chains of HLA-DC molecules result in electrophoretic variants which allow the haplotype of origin of each subunit to be identified by two-dimensional gel electrophoresis. We report here that gene trans-complementation occurs in cells heterozygous for the HLA-D region, resulting in the expression of hybrid HLA-DC molecules in addition to the parental forms. Generation of new HLA-D region hybrid molecules contributes to the qualitative diversity of human MHC class II antigens and has important functional implications in immune regulation.

Oxidized low-density lipoprotein promotes mature dendritic cell transition from differentiating monocyte.

Proinflammatory oxidized phospholipids are generated during oxidative modification of low-density lipoproteins (LDL). The production of these proinflammatory oxidized phospholipids is controlled by secreted enzymes that circulate as proteins complexed with LDL and high-density lipoprotein. During the acute phase response to tissue injury, profound changes occur in lipoprotein enzymatic composition that alter their anti-inflammatory function. Monocytes may encounter oxidized phospholipids in vivo during their differentiation to macrophages or dendritic cells (DC). In this study we show that the presence of oxidized LDL (oxLDL) at the first day of monocyte differentiation to DC in vitro yielded phenotypically atypical cells with some functional characteristics of mature DC. Addition of oxLDL during the late stage of monocyte differentiation gave rise directly to phenotypically mature DC with reduced uptake capacity, secreting IL-12 but not IL-10, and supporting both syngeneic and allogeneic T cell stimulation. In contrast to known mediators of DC activation, oxLDL did not trigger maturation of immature DC. An intriguing possibility is that a burst of oxidized phospholipids is an endogenous activation signal for the immune system, which is tightly controlled by lipoproteins during the acute phase response.

Invariant chain distinguishes between the exogenous and endogenous antigen presentation pathways.

Class I MHC molecules acquire peptides from endogenously synthesized proteins, whereas class II antigens present peptides derived from extracellular compartment molecules. This dichotomy is due to the fact that the invariant chain associates with class II molecules in the endoplasmic reticulum, preventing binding of endogenous peptides. The mutually exclusive binding of peptide and invariant chain to class II molecules suggests that the invariant chain might play a part in autoimmune disease.

Intracellular transport of class II MHC molecules directed by invariant chain.

Three structural motifs in the invariant chain (li) control the intracellular transport of class II major histocompatibility complex molecules. An endoplasmic reticulum retention signal in the full-length li suggests a role for li in the alpha-beta heterodimer assembly. Another signal motif directs a truncated li, alone or associated with individual class II chains, to a degradation compartment by a pathway circumventing the Golgi. When this truncated li binds alpha-beta dimers, a third signal dominates, directing the complex by way of the Golgi to vesicles in the cell periphery, which may represent a subcompartment of recycling endosomes.

Mice lacking the MHC class II-associated invariant chain.

The invariant chain (li) has aroused much interest because of its close association with major histocompatibility complex (MHC) class II molecules. Various functions have been proposed for it; several of these have received experimental support, but most have not been definitively proven, owing largely to uncertainties inherent in the experimental systems employed. We have now generated a line of mice devoid of the invariant chain by introducing a drastic mutation into the li gene. Cells from mutant animals show aberrant transport of MHC class II molecules, resulting in reduced levels of class II complexes at the surface, and these do not have the typical compact conformation indicative of tight peptide binding. Consequently, mutant cells present protein antigens very poorly and mutant mice are deficient in producing and at negatively selecting CD4+ T cells.