Expression profiles of carp IRF-3/-7 correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK1, four pivotal molecules in RIG-I signaling pathway

The cytoplasmic helicase protein RIG-I (retinoic acid-inducible gene I) and downstream signaling molecules, MAVS (mitochondrial antiviral signaling protein), TRAF3 (TNF-receptor-associated factor 3) and TBK1 (TANK-binding kinase 1), have significant roles in the recognition of cytoplasmic 5'-triphosphate ssRNA and short dsRNA, and phosphorylation of IRF-3 (interferon regulatory factor 3) and IRF-7 which is responsible for the induction of type I interferons (IFN). In the present study, the full-length cDNAs of RIG-I, MAVS, TRAF3 and TBK1 were cloned and identified in common carp (Cyprinus carpio L.). The deduced protein of carp RIG-I is of 946 aa (amino acids), consisting of two CARDs (caspase-recruitment domain), a DEXDc (DExD/H box-containing domain), a HELICc (helicase superfamily c-terminal domain) and a RD (regulatory domain). Carp MAVS is of 585 aa, containing a CARD, a proline-rich region and a TM (transmembrane domain). Carp TRAF3 encodes a protein of 573 aa, including a RING (really interesting new gene), two TRAF-type zinc fingers, a coiled coil and a MATH-TRAF3 (meprin and TRAF homology) domain. Carp TBK1 is of 727 aa and contains a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Carp RIG-I, MAVS, TRAF3 and TBK1 mRNAs are ubiquitously expressed in all tissues examined. In response to SVCV infection, carp RIG-I and MAVS mRNAs were up-regulated at different levels in spleen, head kidney and intestine tissues at different time points. Similarly, both carp IRF-3 and IRF-7 mRNAs were significantly up-regulated in the detected tissues. Especially in intestine, the IRF-3 and IRF-7 mRNAs of carp increased and reached 25.3-fold (at 3 dpi) and 224.7-fold (at 5 dpi). Noteworthily, a significant growth of carp TRAF3 and TBK1 mRNA was also mainly found in intestine (7.0-fold and 11.3-fold at 5 dpi, respectively). These data implied that the expression profiles of IRF-3/-7 mRNAs in carp correlate with the up-regulation of RIG-I/MAVS/TRAF3/TBK, and carp RIG-I and MAVS may be involved in antiviral responses through the RIG-I viral recognition signaling pathway in a TRAF3/TBK1-dependent manner.     

Flavivirus induces interferon-beta gene expression through a pathway involving RIG-I-dependent IRF-3 and PI3K-dependent NF-kappaB activation

In this study, we found that infection with flaviviruses, such as Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2), leads to interferon-beta (IFN-beta) gene expression in a virus-replication- and de novo protein-synthesis-dependent manner. NF-kappaB activation is essential for IFN-beta induction in JEV- and DEN-2-infected cells. However, these two viruses seem to preferentially target different members of the interferon regulatory factor (IRF) family. The activation of constitutively expressed IRF-3, characterized by slower gel mobility, dimer formation, and nuclear translocation, is more evident in JEV-infected cells. Other members of the IRF family, such as IRF-1 and IRF-7 are also induced by DEN-2, but not by JEV infection. The upstream molecules responsible for IRF-3 and NF-kappaB activation were further studied. Evidently, a cellular RNA helicase, retinoic acid-inducible gene I (RIG-I), and a cellular kinase, phosphatidylinositol-3 kinase (PI3K), are required for flavivirus-induced IRF-3 and NF-kappaB activation, respectively. Therefore, we suggest that JEV and DEN-2 initiate the host innate immune response through a molecular mechanism involving RIG-I/IRF-3 and PI3K/NF-kappaB signaling pathways.     

抗病毒天然免疫通路中IRF-3调控新机制

干扰素调节因子-3(interferon regulatory factor-3,IRF-3)是IRF家族中重要 转录因子之一,在调控干扰素(interferon, IFN)基因表达和抗病毒天然免疫反应中具有重要作 用. 最新发现的MITA (mediator of IRF-3 activation, 又称STING/ERIS)蛋白是宿主抗病 毒天然免疫反应中的一种重要调节分子. 病毒侵染时,MITA与IRF-3相互作用,特异性激活 IRF-3,并募集TANK结合激酶1（TANK binding kinase 1, TBK1）与IFN通路中的线粒体抗 病毒信号蛋白MAVS(mitochondrial anti-viral signaling protein)形成复合物,且MITA可 被TBK1磷酸化,诱导Ⅰ型IFN及IFN刺激基因(interferon stimulate genes, ISG)的表达 ,诱发抗病毒天然免疫反应. 同时还发现,泛素连接酶RNF5（ring finger protein 5）可对MITA 发生泛素化修饰从而抑制其对IRF-3活化,实现对宿主抗病毒天然免疫反应负调节作用. 本 室研究发现,严重性急性呼吸系统综合症冠状病毒(severe acute respiratory syndrome co ronavirus, SARS-CoV）和人类新型冠状病毒（human coronavirus NL63, HCoV-NL63）的 木瓜样蛋白酶（papain-like protease, PLP）利用其特有的去泛素化酶（deubiquitinase, DUB）活性,通过宿主细胞泛素-蛋白酶体信号系统对IRF-3的泛素化等翻译后修饰进行调节 ,从而成为该种病毒逃逸机体抗病毒防御系统主要手段之一.     

Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated?

The IkappaB kinases (IKKs) IKK-alpha and IKK-beta, and the IKK-related kinases TBK1 and IKK-epsilon, have essential roles in innate immunity through signal-induced activation of NF-kappaB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kappaB essential modulator coordinates some IKK complexes, whereas TANK, NF-kappaB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-epsilon complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-epsilon subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity.     

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.     

Differential activation of IFN regulatory factor (IRF)-3 and IRF-5 transcription factors during viral infection

Members of the IFN regulatory factor (IRF) family regulate gene expression critical to immune response, hemopoiesis, and proliferation. Although related by homology at their N-terminal DNA-binding domain, they display individual functional properties. The distinct properties result from differences in regulated expression, response to activating signals, and interaction with DNA regulatory elements. IRF-3 is expressed ubiquitously and is activated by serine phosphorylation in response to viral infection or TLR signaling. Evidence indicates that the kinases TANK-binding kinase 1 and inhibitor of NF-kappaB kinase-epsilon specifically phosphorylate and thereby activate IRF-3. We evaluated the contribution of another member of the IRF family, IRF-5, during viral infection since prior studies provided varied results. Analysis of phosphorylation, nuclear translocation, dimerization, binding to CREB-binding protein, recognition of DNA, and induction of gene expression were used comparatively with IRF-3 as a measure of IRF-5 activation. IRF-5 was not activated by viral infection; however, expression of TANK-binding kinase 1 or inhibitor of NF-kappaB kinase-epsilon did provide clear activation of IRF-5. IRF-5 is therefore distinct in its activation profile from IRF-3. However, similar to the biological effects of IRF-3 activation, a constitutively active mutation of IRF-5 promoted apoptosis. The apoptosis was inhibited by expression of Bcl-x(L) but not a dominant-negative mutation of the Fas-associated death domain. These studies support the distinct activation profiles of IRF-3 in comparison to IRF-5, but reveal a potential shared biological effect.     

Activation of an immunoregulatory and antiviral gene expression program in poly(I:C)-transfected human neutrophils

Neutrophils, historically known for their involvement in acute inflammation, are also targets for infection by many different DNA and RNA viruses. However, the mechanisms by which they recognize and respond to viral components are poorly understood. Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic mimetic of viral dsRNA that is known to interact either with endosomal TLR3 (not expressed by human neutrophils) or with cytoplasmic RNA helicases such as melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene I (RIG-I). In this study, we report that intracellularly administered poly(I:C) stimulates human neutrophils to specifically express elevated mRNA levels encoding type I IFNs, immunoregulatory cytokines, and chemokines, such as TNF-alpha, IL-12p40, CXCL10, CXCL8, CCL4, and CCL20, as well as classical IFN-responsive genes (IRG), including IFIT1 (IFN-induced protein with tetratricopeptide repeats 1)/IFN-stimulated gene (ISG)56, G1P2/ISG15, PKR (dsRNA-dependent protein kinase), and IFN-regulatory factor (IRF)7. Investigations into the mechanisms whereby transfected poly(I:C) promotes gene expression in neutrophils uncovered a crucial involvement of the MAPK-, PKR-, NF-kappaB-, and TANK (TNF receptor-associated NF-kappaB kinase)-binding kinase (TBK1)/IRF3-signaling transduction pathways, as illustrated by the use of specific pharmacological inhibitors. Consistent with the requirement of the cytoplasmic dsRNA pathway for antiviral signaling, human neutrophils were found to constitutively express significant levels of both MDA5 and RIG-I, but not TLR3. Accordingly, neutrophils isolated from MDA5-deficient mice had a partial impairment in the production of IFN-beta and TNF-alpha upon infection with encephalomyocarditis virus. Taken together, our data demonstrate that neutrophils are able to activate antiviral responses via helicase recognition, thus acting at the frontline of immunity against viruses.