Françoise Mégret

Virus Infection Switches TLR-3-Positive Human Neurons To Become Strong Producers of Beta Interferon

ABSTRACT To study the capacity of human neurons to mount innate immunity responses to viral infections, we infected cells of a human postmitotic neuron-derivative cell line, NT2-N, with rabies virus (RABV) and herpes simplex type 1 (HSV-1). Changes in neuronal gene expression were analyzed by use of Affymetrix microarrays. Applying a twofold cutoff, RABV increased the transcription of 228 genes, and HSV-1 increased the transcription of 263 genes. The most striking difference between the two infections concerns genes involved in immunity. These genes represent 24% of the RABV-upregulated genes and only 4.9% of the HSV-1-upregulated genes. Following RABV infection, the most upregulated genes belong to the immunity cluster and included almost exclusively genes for beta interferon (IFN-β) primary and secondary responses as well as genes for chemokines (CCL-5, CXCL-10) and inflammatory cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha, interleukin 1 alpha). In contrast, HSV-1 infection did not increase IFN-β gene transcripts and triggered the production of only IL-6 and interferon regulatory factor 1 mRNAs. The microarray results were confirmed by real-time PCR, immunocytochemistry, and enzyme-linked immunosorbent assay. Human neurons were found to express Toll-like receptor 3. They produced IFN-β after treatment with poly(I:C) but not with lipopolysaccharide. Thus, human neurons can mount an innate immunity response to double-stranded RNA. These observations firmly establish that human neurons, in absence of glia, have the intrinsic machinery to sense virus infection.

The innate immune facet of brain: human neurons express TLR-3 and sense viral dsRNA.

Inflammation is an important factor in the pathogenesis of neurodegenerative diseases, such as Alzheimers disease or multiple sclerosis, and during microbial infections of the nervous system. Glial cells were thought to be the main contributor for cytokine and chemokine production and Toll-like receptor (TLR) expression in the brain. Here, we report that human neurons express TLR-3, a major receptor in virus-mediated innate immune response. We established that these cells can mount a strong inflammatory response characterized by the expression of inflammatory cytokines (TNF-α, IL-6), chemokines (CCL-5 and CXCL-10), and antiviral molecules (2′5′OAS and IFN-β) after treatment with dsRNA—a by-product of viral infection and ligand of TLR-3. This work firmly establishes that human neurons, in absence of glia, have the intrinsic machinery to trigger robust inflammatory, chemoattractive, and antiviral responses.

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism

Dengue virus infections are a growing public health concern and strategies to control the spread of the virus are urgently needed. The murine monoclonal antibody 4E11 might be of interest, since it neutralizes dengue viruses of all serotypes by binding to the 296-400 segment of the major dengue virus envelope glycoprotein (DE). When phage-displayed peptide libraries were screened by affinity for 4E11, phage clone C1 was selected with a 50% frequency. C1 shared three of nine residues with DE(306-314) and showed significant reactivity to 4E11 in ELISA. C1-induced antibodies cross-reacted with DE(296-400) in mice, suggesting that it was a structural equivalent of the native epitope of 4E11 on DE. Accordingly, 4E11 bound to the DE(306-314) synthetic peptide and this reaction was inhibited by DE(296-400). Moreover, DE(306-314) could block dengue virus infection of target cells in an in vitro assay. A three-dimensional model of DE revealed that the three amino acids shared by DE(296-400) and C1 were exposed to the solvent and suggested that most of the amino acids comprising the 4E11 epitope were located in the DE(306-314) region. Since 4E11 blocked the binding of DE(296-400) to heparin, which is a highly sulfated heparan sulfate (HSHS) molecule, 4E11 may act by neutralizing the interaction of DE(306-314) with target cell-displayed HSHS. Our data suggest that the DE(306-314) segment is critical for the infectivity of all dengue virus serotypes and that molecules that block the binding of DE(306-314) to HSHS may be antiviral reagents of therapeutic interest.

Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein

Dengue virus (DENV) causes the major arboviral disease of the tropics, characterized in its severe forms by signs of hemorrhage and plasma leakage. DENV encodes a nonstructural glycoprotein, NS1, that associates with intracellular membranes and the cell surface. NS1 is eventually secreted as a soluble hexamer from DENV-infected cells and circulates in the bloodstream of infected patients. Extracellular NS1 has been shown to modulate the complement system and to enhance DENV infection, yet its structure and function remain essentially unknown. By combining cryoelectron microscopy analysis with a characterization of NS1 amphipathic properties, we show that the secreted NS1 hexamer forms a lipoprotein particle with an open-barrel protein shell and a prominent central channel rich in lipids. Biochemical and NMR analyses of the NS1 lipid cargo reveal the presence of triglycerides, bound at an equimolar ratio to the NS1 protomer, as well as cholesteryl esters and phospholipids, a composition evocative of the plasma lipoproteins involved in vascular homeostasis. This study suggests that DENV NS1, by mimicking or hijacking lipid metabolic pathways, contributes to endothelium dysfunction, a key feature of severe dengue disease.

Modulation of HLA-G Expression in Human Neural Cells after Neurotropic Viral Infections

ABSTRACT HLA-G is a nonclassical human major histocompatibility complex class I molecule. It may promote tolerance, leading to acceptance of the semiallogeneic fetus and tumor immune escape. We show here that two viruses—herpes simplex virus type 1 (HSV-1), a neuronotropic virus inducing acute infection and neuron latency; and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection—upregulate the neuronal expression of several HLA-G isoforms, including HLA-G1 and HLA-G5, the two main biologically active isoforms. RABV induces mostly HLA-G1, and HSV-1 induces mostly HLA-G3 and HLA-G5. HLA-G expression is upregulated in infected cells and neighboring uninfected cells. Soluble mediators, such as beta interferon (IFN-β) and IFN-γ, upregulate HLA-G expression in uninfected cells. The membrane-bound HLA-G1 isoform was detected on the surface of cultured RABV-infected neurons but not on the surface of HSV-1-infected cells. Thus, neuronotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially. HLA-G may therefore be involved in the escape of certain viruses from the immune response in the nervous system.

Toll-Like Receptor 3 (TLR3) Plays a Major Role in the Formation of Rabies Virus Negri Bodies

Human neurons express the innate immune response receptor, Toll-like receptor 3 (TLR3). TLR3 levels are increased in pathological conditions such as brain virus infection. Here, we further investigated the production, cellular localisation, and function of neuronal TLR3 during neuronotropic rabies virus (RABV) infection in human neuronal cells. Following RABV infection, TLR3 is not only present in endosomes, as observed in the absence of infection, but also in detergent-resistant perinuclear inclusion bodies. As well as TLR3, these inclusion bodies contain the viral genome and viral proteins (N and P, but not G). The size and composition of inclusion bodies and the absence of a surrounding membrane, as shown by electron microscopy, suggest they correspond to the previously described Negri Bodies (NBs). NBs are not formed in the absence of TLR3, and TLR3−/− mice—in which brain tissue was less severely infected—had a better survival rate than WT mice. These observations demonstrate that TLR3 is a major molecule involved in the spatial arrangement of RABV–induced NBs and viral replication. This study shows how viruses can exploit cellular proteins and compartmentalisation for their own benefit.

Dengue 1 virus binding to human hepatoma HepG2 and simian Vero cell surfaces differs

We analysed the binding and infectivity of dengue virus serotype 1 (DEN-1) for the human hepatoma cell line HepG2 in comparison with the simian kidney cell line Vero. The higher susceptibility of Vero cells to DEN-1 correlated with greater binding affinity of DEN-1 to these cells. In contrast, the capacity of virus attachment was higher for HepG2 than for Vero cells. Profiles of DEN-1 binding at different pH were markedly different between the two cell types. A type-specific neutralizing monoclonal antibody reduced initial virus binding to both cell types similarly but complex- and group-specific neutralizing antibodies affected virus adhesion differently. Altogether, these results suggest the involvement of different receptors or receptors presented in a different environment on the cell surface in the two cell lines. The sensitivity to proteolytic enzymes and to ionic detergent of the binding sites on the two cell types was tested and results indicated that they may be multimeric proteins or protein complexes.

Detrimental Contribution of the Immuno-Inhibitor B7-H1 to Rabies Virus Encephalitis

Rabies virus is the etiological agent of an acute encephalitis, which in absence of post exposure treatment is fatal in almost all cases. Virus lethality rests on its ability to evade the immune response. In this study, we analyzed the role of the immuno-inhibitory molecule B7-H1 in this virus strategy. We showed that in the brain and spinal cord of mice, rabies virus infection resulted in significant up-regulation of B7-H1 expression, which is specifically expressed in infected neurons. Correlatively, clinical rabies in B7-H1−/− mice is markedly less severe than in wild-type mice. B7-H1−/− mice display resistance to rabies. Virus invasion is reduced and the level of migratory CD8 T cells increases into the nervous system, while CD4/CD8 ratio remains unchanged in the periphery. In vivo, neuronal B7-H1 expression is critically depending on TLR3 signaling and IFN-β, because TLR3−/− mice—in which IFN-β production is reduced—showed only a limited increase of B7-H1 transcripts after infection. These data provide evidence that neurons can express the B7-H1 molecule after viral stress or exposure to a particular cytokine environment. They show that the B7-H1/PD-1 pathway can be exploited locally and in an organ specific manner—here the nervous system—by a neurotropic virus to promote successful host invasion.

Attenuation of Rabies Virulence: Takeover by the Cytoplasmic Domain of Its Envelope Protein

Survival of rabies virus–infected neurons depends on a single amino acid in the PDZ-binding site of a viral protein. Tipping the Balance Strains of rabies virus, which infects neurons, may be virulent, in which case the cells survive long enough for the virus to replicate and spread, or they may be attenuated, in which case the infected cells die by apoptosis. Préhaud et al. compared one attenuated and one virulent viral strain and found that a single amino acid change in a region of a viral envelope protein that binds to host cell proteins was sufficient to account for the death or survival of infected cells. The binding properties of the attenuated virus protein were expanded, thereby affecting the balance in the activities of host kinases and phosphatases sufficiently to trigger cell death. These findings may inform strategies to engineer attenuated viruses, which are often used in live vaccines. The capacity of a rabies virus to promote neuronal survival (a signature of virulence) or death (a marker of attenuation) depends on the cellular partners recruited by the PDZ-binding site (PDZ-BS) of its envelope glycoprotein (G). Neuronal survival requires the selective association of the PDZ-BS of G with the PDZ domains of two closely related serine-threonine kinases, MAST1 and MAST2. Here, we found that a single amino acid change in the PDZ-BS triggered the apoptotic death of infected neurons and enabled G to interact with additional PDZ partners, in particular the tyrosine phosphatase PTPN4. Knockdown of PTPN4 abrogated virus-mediated apoptosis. Thus, we propose that attenuation of rabies virus requires expansion of the set of host PDZ proteins with which G interacts, which interferes with the finely tuned homeostasis required for survival of the infected neuron.