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.

Ambivalent role of the innate immune response in rabies virus pathogenesis.

The neurotropic rabies virus (RABV) has developed several evasive strategies, including immunoevasion, to successfully infect the nervous system (NS) and trigger a fatal encephalomyelitis. Here we show that expression of LGP2, a protein known as either a positive or negative regulator of the RIG-I-mediated innate immune response, is restricted in the NS. We used a new transgenic mouse model (LGP2 TG) overexpressing LGP2 to impair the innate immune response to RABV and thus revealed the role of the RIG-I-mediated innate immune response in RABV pathogenesis. After infection, LGP2 TG mice exhibited reduced expression of inflammatory/chemoattractive molecules, beta interferon (IFN-ß), and IFN-stimulated genes in their NS compared to wild-type (WT) mice, demonstrating the inhibitory function of LGP2 in the innate immune response to RABV. Surprisingly, LGP2 TG mice showed more viral clearance in the brain and lower morbidity than WT mice, indicating that the host innate immune response, paradoxically, favors RABV neuroinvasiveness and morbidity. LGP2 TG mice exhibited similar neutralizing antibodies and microglia activation to those of WT mice but showed a reduction of infiltrating CD4(+) T cells and less disappearance of infiltrating CD8(+) T cells. This occurred concomitantly with reduced neural expression of the IFN-inducible protein B7-H1, an immunoevasive protein involved in the elimination of infiltrated CD8(+) T cells. Our study shows that the host innate immune response favors the infiltration of T cells and, at the same time, promotes CD8(+) T cell elimination. Thus, to a certain extent, RABV exploits the innate immune response to develop its immunoevasive strategy.

Attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein.

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.

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.

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-beta, because TLR3(-/-) mice--in which IFN-beta 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.

Modulation of HLA-G and HLA-E expression in human neuronal cells after rabies virus or herpes virus simplex type 1 infections.

Human Leukocyte Antigen (HLA)-G and E are nonclassical human MHC class I molecules. They may promote tolerance leading to virus and tumor immune escape. We recently described that the herpes simplex virus type 1 (HSV-1), a neurotropic virus inducing chronic infection and neuron latency, and rabies virus (RABV), a neuronotropic virus triggering acute neuron infection, up-regulate HLA-G expression in human neurons (NT2-N). Surface expression was only detected after RABV infection. We investigated here whether RABV and HSV-1 up-regulate HLA-E expression in human neuronal precursors (Ntera-2D/1). We found that RABV, not HSV-1, up-regulates HLA-E expression, nevertheless HLA-E could not be detected on the surface of RABV-infected Ntera-2D/1. Altogether these data suggest that HLA-G and not HLA-E could contribute to the immune escape of RABV. In contrast, there was no evidence that these molecules are used by latent HSV-1 infection. Thus, neurotropic viruses that escape the host immune response totally (RABV) or partially (HSV-1) regulate HLA-G expression on human neuronal cells differentially.

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 Alzheimer's 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-alpha, IL-6), chemokines (CCL-5 and CXCL-10), and antiviral molecules (2'5'OAS and IFN-beta) 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.

Modulation of HLA-G expression in human neural cells after neurotropic viral infections.

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-beta) and IFN-gamma, 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.

Virus infection switches TLR-3-positive human neurons to become strong producers of beta interferon.

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-beta) 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-beta 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-beta 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.

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.