A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome

IFN-Lambda (IFN-λ) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-α/β (type I IFN) and IFN-λ (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-α/β and IFN-λ systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-λ. In the brain, IFN-α/β was readily produced after infection with various RNA viruses, whereas expression of IFN-λ was low in this organ. In the liver, virus infection induced the expression of both IFN-α/β and IFN-λ genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-α/β and IFN-λ to be compared. The response to IFN-λ correlated with expression of the α subunit of the IFN-λ receptor (IL-28Rα). The IFN-λ response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-λ in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-α/β was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-λ system probably evolved to specifically protect epithelia. IFN-λ might contribute to the prevention of viral invasion through skin and mucosal surfaces

Analyse de l'activité différentielle et de la propagation, in vivo, des interférons de type I et de type III

Type I interferons (IFNs- / ) are a large family of cytokines which play a crucial role in the antiviral defense. They also contribute to the control of the immune and antitumoral responses. Important divergences exist between the sequences of different members of the type I IFN family. Moreover, some IFNs- / are glycosylated and some are not. In spite their differences, all type I IFN subtypes bind the same receptor and induce similar biological responses. This raises the question of the reason for the multiplicity of IFNs- / genes. Recently, a new IFN family was discovered and called type III IFNs (or IFNs- ). Type III IFNs bind a receptor distinct from that of type I IFNs but seem to induce very similar antiviral and antiproliferative effects. Moreover, type III IFNs are produced in response to the same stimuli as type I IFNs. The aim of this work was to understand to what extent various IFNs are redundant in their functions in vivo. First, we analyzed the impact of glycosylation on IFN activity. We observed that murine IFN- carries 3 glycosylation sites. We showed that, in vitro, the complete loss of glycosylation induced a dramatic decrease of IFN- antiviral activity. This decrease is probably due to poor solubility of the non-glycosylated form of the IFN, as reported for human IFN- . To test the impact of glycosylation in vivo, we compared the activities of the non-glycosylated IFN- 6T and of a mutant of this IFN in which a glycosylation site was added by mutagenesis. Both IFNs presented a similar antiviral activity, in vitro. When expressed in vivo, by electroinjection of expression plasmids, glycosylated and non glycosylated IFN- 6T could induce ISGs expression in all peripheral organs examined and in the CNS. However, we failed to detect any impact of glycosylation on IFN activity, in vivo. Then, we compared, in vivo, the responses to circulating type I and to type III IFNs, in several organs, using the same expression protocol. In contrast to type I IFNs, type III IFNs induced a response that was highly tissue-specific. At the cellular level, we observed that this response was specific to epithelial cells whereas cellular response to type I IFNs occurred in many cell types and was prominent in the endothelial cells. This work provides some evidence for partial divergence between the functions of type I and type III IFNs. Yet, much has to be learned to understand the reason of the multiplicity of IFNs genes and of the coexistence of the type I and type III IFN system.Les interférons (IFNs) de type I (ou IFNs- / ) constituent une famille multigénique de cytokines qui possèdent des fonctions cruciales dans la défense de l'organisme face aux infections virales. En outre, les IFNs- / exercent une influence importante sur le développement de la réponse immunitaire acquise et possèdent des propriétés antitumorales. Il existe des divergences importantes entre les séquences des différents IFNs de type I (IFN- , IFN- , IFN- ,). De plus, certains IFNs sont glycosylés et d'autres ne le sont pas. Malgré leurs différences, tous ces IFNs se lient au même récepteur cellulaire et exercent des activités très similaires, ce qui soulève la question de la raison de la multiplicité des gènes codant les IFNs. Récemment, une nouvelle famille d'IFNs a été identifiée, constituée des interleukines IL-28 et IL-29 (appelées aussi IFNs- ou IFNs de type III). Bien que les IFNs- se lient à un récepteur différent de celui des IFNs de type I, ils induisent des effets antiviraux et antiprolifératifs étonnamment similaires à ceux développés par les IFNs- / . Les IFNs de type I et de type III sont produits en réponse aux mêmes stimuli et induisent l'activation des mêmes voies de signalisation. Le but de ce travail était d'analyser dans quelle mesure les différents sous-types d'IFN se distinguent par la spécificité de leur distribution et de leur activité, in vivo. Dans un premier temps, nous avons analysé l'influence de la glycosylation sur l'activité des IFNs. Nous avons observé que l'IFN- murin possède trois sites de glycosylation, dont un est situé à proximité du site de fixation au récepteur. Nous avons montré que, in vitro, l'absence complète de glycosylation de cet IFN altère fortement son activité, probablement en raison d'une instabilité de la molécule. Pour tester l'influence de la glycosylation des IFNs in vivo, nous avons utilisé l'IFN- 6T. Un mutant (D78N) de cet IFN pourvu d'un site de glycosylation montre, in vitro, une activité biologique comparable à celle de l'IFN- 6T sauvage (non glycosylé). En utilisant un protocole d'électro-injection intra-musculaire de plasmides d'expression, nous avons observé que, in vivo, l'IFN- 6T glycosylé et non glycosylé induisaient l'activation de l'expression d'ISGs dans tous les organes périphériques ainsi que dans le SNC. Cependant, nous n'avons pas observé d'effet de la glycosylation sur l'activité de l'IFN in vivo. Nous avons ensuite comparé, en utilisant le même protocole d'expression in vivo, la réponse différentielle des différents organes aux IFNs circulants, de type I et de type III. Contrairement aux IFNs de type I, les IFN de type III induisent une réponse restreinte à certains tissus. Au niveau cellulaire, la réponse aux IFNs de type III semble spécifique des cellules epithéliales alors que la réponse aux IFNs de type I est majoritairement due aux cellules endothéliales. Enfin, dans un modèle viral de neuroinvasion, nous avons observé que l'IFN- avait peu d'effet protecteur, contrairement à l'IFN- . La différence de réponse aux IFNs de type I et de type III donne un nouvel éclairage dans la compréhension de la coexistence de 2 systèmes qui, à première vue, semblent redondantsThèse de doctorat en sciences biomédicales (immuno-virologie) (SBIM 3)--UCL, 200

N-glycosylation of murine IFN-beta in a putative receptor-binding region.

Human and mouse genomes contain more than 20 related genes encoding diverse type I interferons (IFNs- alpha/beta), cytokines that are crucial for resistance of organisms against viral infections. Although the amino acid sequences of various IFN-alpha/beta subtypes differ markedly, they are all considered to share a common three-dimensional structure and to bind the same heterodimeric receptor, composed of the IFNAR-1 and IFNAR-2 subunits. Analysis of available mammalian IFN-beta sequences showed that they all carry 1 to 5 predicted N-glycosylation sites. Murine IFN-beta contains three predicted N-glycosylation sites (Asn29, Asn69, Asn76), one of which (Asn29) is located in the AB loop, in a region predicted to interact with the type I IFN receptor. The aim of this work was to test if this site is indeed N-glycosylated and if this glycosylation would affect IFN antiviral activity. We showed that all three N-glycosylation sites predicted from the sequence, including Asn29, carry N-linked sugars. Mutation of individual N-glycosylation sites had a weak negative influence on IFN antiviral activity. In contrast, the complete loss of glycosylation dramatically decreased activity. Our data suggest that interaction of murine IFN-beta with the IFNAR could locally differ from that of human IFN-alpha2 and human IFN-beta

Type I interferon response in the central nervous system.

This review is dedicated to the influence of type I IFNs (also called IFN-alpha/beta) in the central nervous system (CNS). Studies in mice with type I IFN receptor or IFN-beta gene deficiency have highlighted the importance of the type I IFN system against CNS viral infections and non-viral autoimmune disorders. Direct antiviral effects of type I IFNs appear to be crucial in limiting early spread of a number of viruses in CNS tissues. Type I IFNs have also proved to be beneficial in autoimmune disorders like multiple sclerosis or experimental autoimmune encephalitis, probably through immunomodulatory effects. Increasing efforts are done to characterize IFN expression and response in the CNS: to identify type I IFN producing cells, to decipher pathways leading to type I IFN expression in those cells, and to identify responding cells. However, reversible and irreversible damages consecutive to chronic exposure of the CNS to type I IFNs underline the importance of a tightly regulated type I IFN homeostasis in this organ

Anti-IL-17A autovaccination prevents clinical and histological manifestations of experimental autoimmune encephalomyelitis.

Excessive or inappropriate production of IL-17A has been reported in diseases such as rheumatoid arthritis, asthma, and multiple sclerosis. The potential clinical relevance of these correlations was suggested by the protective effects of anti-IL-17A monoclonal antibodies in various mouse disease models. However, the chronic nature of the corresponding human afflictions raises great challenges for Ab-based therapies. An alternative to passive Ab therapy is autovaccination. Covalent association of self-cytokines with foreign proteins has been reported to induce the production of antibodies capable of neutralizing the biological activity of the target cytokine. We recently reported that cross-linking of IL-17A to ovalbumin produced highly immunogenic complexes that induced long-lasting IL-17A-neutralizing antibodies. Vaccinated SJL mice were completely protected against experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein peptide (PLP 139-151), and a monoclonal anti-IL-17A Ab (MM17F3), derived from C57Bl/6 mice vaccinated against IL-17A-OVA, also prevented disease development. Here we report that this Ab also protects C57Bl/6 mice from myelin oligdendrocyte glycoprotein (MOG)-induced EAE. Histological analysis of brain sections of C57Bl/6 mice treated with MM17F3 showed a complete absence of inflammatory infiltrates and evidence for a marked inhibition of chemokine and cytokine messages in the spinal cord. These results further extend the analytical and therapeutic potential of the autovaccine procedure

Role of the interleukin (IL)-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/interferon-lambda 1 - Similarities with type I interferon signaling

Interferon (IFN)-lambda1, -lambda2, and -lambda3 are the latest members of the class II cytokine family and were shown to have antiviral activity. Their receptor is composed of two chains, interleukin-28R/likely interleukin or cytokine or receptor 2 (IL-28R/LICR2) and IL-10Rbeta, and mediates the tyrosine phosphorylation of STAT1, STAT2, STAT3, and STAT5. Here, we show that activation of this receptor by IFN-lambda1 can also inhibit cell proliferation and induce STAT4 phosphorylation, further extending functional similarities with type I IFNs. We used IL-28R/LICR2-mutated receptors to identify the tyrosines required for STAT activation, as well as antiproliferative and antiviral activities. We found that IFN-lambda1-induced STAT2 tyrosine phosphorylation is mediated through tyrosines 343 and 517 of the receptor, which showed some similarities with tyrosines from type I IFN receptors involved in STAT2 activation. These two tyrosines were also responsible for antiviral and antiproliferative activities of IFN-lambda1. By contrast, STAT4 phosphorylation ( and to some extent STAT3 activation) was independent from IL-28R/LICR2 tyrosine residues. Taken together, these observations extend the functional similarities between IFN-lambdas and type I IFNs and shed some new light on the mechanisms of activation of STAT2 and STAT4 by these cytokines

IFN-α and IFN-λ responding cells in the kidney adipose tissue and in the brain.

<p>Mx1 expression, detected by immunohistochemistry (as white nuclear spots), 7 days after electroinjection of a plasmid coding for MuIFN-α6T or MuIFN-λ3. A–C–E: Mx1/WT mouse electroinjected with a plasmid coding for IFN-α6T. B–D–F: Mx1/IFNAR1-KO mouse electroinjected with a plasmid coding for IFN-λ3. A–B: sections showing the kidney adipose tissue. C–D: brain sections showing the choroid plexus of the 4th ventricle. E–F: Higher magnification of the choroid plexus. G: Cartoon showing the structural organization of the choroid plexus.</p