【作者】 赵凯；

【导师】 高成江；

【作者基本信息】 山东大学 ， 免疫学， 2016， 博士

【摘要】 固有免疫系统作为机体抵御外界病原体入侵的首道防线,不仅可以激活适应性免疫系统,同时也可以直接对入侵的病原体产生强烈的免疫应答,从而杀伤病原体。在病毒入侵时,固有免疫系统可以激活产生多种细胞因子,其中最主要的是I型干扰素(IFNα/β)。固有免疫系统中的多种的模式识别受体(PRRs)都与Ⅰ型干扰素的产生相关,包括诸如Toll样受体(TLRs), RIG-I样受体(RLRs)和内源性DNA感受器等。产生的Ⅰ型干扰素可以进而与其受体结合,活化JAK (Janus kinase)和STAT (Signaltransducers and activators of transcription)信号通路,进而诱导IFN-stimulated genes (ISGs)的表达,最终清除病毒。骨桥蛋白(osteopontin, OPN)又称为早期T淋巴细胞活化因子1(early T lymphocyte activation 1, Etal),是一种分泌性的多功能糖蛋白。OPN可以调节多种生物体进程,包括细胞分化、粘附、骨重构、恶性肿瘤和免疫反应。长期以来,OPN被认为是一个与炎症过程有关的潜在的促炎症细胞因子,具有促进巨噬细胞分泌IFN-γ和IL-12的作用。近年来,随着胞内形式OPN (iOPN)的发现,OPN在不同免疫细胞以及免疫反应的不同阶段中的不同调控作用逐渐引起人们的重视。而iOPN在固有免疫系统当中尤其是抗病毒免疫当中的作用还未明确。研究目的：探讨OPN,尤其是iOPN是否可以调节病毒诱导的Ⅰ型干扰素产生。如若可以调节Ⅰ型干扰素产生,阐述其潜在机制。研究方法：1.首先检测不同病毒及刺激剂对OPN表达的影响。再利用OPN缺陷型(Spp1-/-)小鼠与野生型(WT)小鼠的腹腔巨噬细胞,经病毒刺激后,检测IFNβ以及下游多种代表性ISGs的mRNA水平变化、蛋白分泌水平变化等。通过构建全长型OPN (full length OPN)及胞内形式OPN(iOPN)表达质粒并转染进HEK293细胞,利用双荧光素酶报告基因,检测两种不同形式OPN对于调节RNA病毒SeV所活化的IFN表达的影响,并检测iOPN质粒对于各种接头分子所活化的IFN启动子区活性的调节作用。2.利用OPN缺陷型(Sppl-/-)小鼠与野生型(WT)小鼠,通过腹腔注射VSV病毒,检测VSV病毒在小鼠脏器中的滴度与含量,反应两类小鼠对于VSV病毒的抵抗能力的区别。3.通过利用OPN缺陷型(Spp1-/-)小鼠与野生型(WT)小鼠腹腔巨噬细胞,以及iOPN过表达质粒转染HEK293细胞,检测经SeV刺激后,重要的IFN转录因子IRF3的活化水平变化。4.经实时荧光定量PCR (q-PCR)以及双荧光素酶报告基因实验,检测iOPN质粒的靶点分子,并经免疫共沉淀(co-IP)以及免疫荧光等实验检测iOPN与靶点分子的结合情况。5.明确靶点分子后,利用不同类型的泛素分子质粒,共转染iOPN及靶点分子进HEK293细胞,检测其是否发生泛素化水平的变化,以及靶点分子的稳定性是否发生变化。6.靶点分子若发生K48位泛素化水平的变化以及其稳定性若发生变化,说明iOPN可能可以调节靶点分子的稳定性。通过对靶点已知的泛素化酶以及去泛素化酶进行筛选,寻找可能的作用机制,并通过细胞水平以及体外翻译系统对结论进行实验性确认。7.通过利用细胞过表达载体以及慢病毒表达载体构建iOPN-WT、iOPN-N、iOPN-C重组过表达载体,通过体外翻译系统实验或对OPN缺陷型小鼠腹腔巨噬细胞进行iOPN回补实验,明确iOPN具体发生功能的区域。研究结果：1.首先,在利用RNA病毒VSV(水泡性口炎病毒)、SeV(仙台病毒)以及DNA病毒HSV(单纯疱疹病毒)等病毒刺激野生型小鼠腹腔巨噬细胞不同时间点后,发现OPN的表达都有逐渐升高的趋势；其次,在OPN缺陷型小鼠以及野生型小鼠巨噬细胞中加入SeV或VSV刺激不同时间后,发现相比较于野生型组,OPN缺陷型小鼠腹腔巨噬细胞产生IFNβ的能力都明显降低；将构建成功的全长型OPN表达载体与iOPN表达载体分别转染进HEK293后经SeV刺激后,发现二种质粒都可以使IFNβ表达增强,但是iOPN表达载体组明显强于全长型OPN表达载体组,并且利用OPN封闭抗体封闭分泌型OPN后,IFNβ的产生无明显变化,说明全长型OPN所表达的分泌性OPN可能并不参与调节IFNβ的增强过程；iOPN也可以明显增强RIG-I、MDA5、TRIF以及STING&cGAS等接头分子所介导的IFNβ启动子区的活化。2.OPN缺陷型小鼠相比较于野生型小鼠,会产生较少的IFNβ,进而造成此组巨噬细胞或成年小鼠在经VSV病毒感染后,VSV病毒的滴度以及VSV mRNA水平以及VSV病毒的蛋白表达程度更高。并且OPN缺陷型小鼠组在大剂量VSV病毒感染后,生存率明显低于野生型小鼠组。3.前述实验结果证明OPN,尤其是iOPN的确可以正调IFNβ的产生,又由于IRF3是重要的调控IFNβ表达的转录因子,我们进而研究IRF3的活化水平是否有变化。我们发现无论是在启动子区结合、磷酸化、二聚化以及入核等方面,iOPN都可以起到正调作用,说明iOPN的确是通过影响IRF3转录因子的活化进而增强了IFNβ的表达。4.为了明确iOPN作用的靶点,我们经报告基因以及realtime-PCR等技术发现iOPN作用的靶点可能存在于MAVS以及TBKl附近。通过免疫共沉淀我们发现iOPN可以特异性与TRAF3结合,并且这一结论也经免疫荧光以及体外蛋白结合实验验证。5.在知道作用靶点为TRAF3后,我们进一步研究可能的机制。泛素化调节作用在机体中具有举足轻重的作用,我们首先研究下iOPN是否会影响TRAF3的泛素化。结果发现,iOPN可以抑制TRAF3发生K48位泛素化修饰,即iOPN可以起到稳定TRAF3表达的作用。无论是在HEK293细胞中过表达iOPN,或是在OPN缺陷型的小鼠腹腔巨噬细胞,iOPN的过表达或缺失都会导致放线菌酮(CHX)介导的TRAF3的稳定性发生明显变化。6.由于我们发现iOPN可以抑制TRAF3发生K48位泛素化修饰,但由于iOPN本身并没有去泛素化酶功能,这就提示我们可能iOPN可以抑制某种可介导TRAF3的K48位泛素化作用的E3连接酶与其结合,或者募集某种可以去除TRAF3的K48位泛素化链的去泛素化酶与TRAF3结合,结果我们发现之前有报道过的TRAF3的去泛素化酶USP25并没有因为iOPN的加入而更加明显的除去TRAF3上的泛素化链,而Triad3A对于TRAF3的K48位泛素化修饰作用则由于iOPN的加入而明显受到抑制。进而我们发现iOPN可以明显的抑制TRAF3与Triad3A的结合,并且TRAF3的Y440、Q442位点如若被突变则TRAF3不与Triad3A结合,同时也不与iOPN结合。综上说明iOPN可能是通过结合了TRAF3进而抑制了Triad3A与TRAF3的结合,iOPN与Triad3A肯能是竞争结合关系。7.通过构建的iOPN-WT以及iOPN-N端或iOPN-C端截短体,我们发现iOPN的C端可以与TRAF3发生结合,并且通过在OPN缺陷型小鼠腹腔巨噬细胞中进行iOPN回补实验,我们进一步确定iOPN的C端即足以发挥结合TRAF3、抑制TRAF3的K48位泛素化以及增强IFNβ产生的作用。总结：1.OPN的表达可被病毒所诱导,并且OPN尤其是iOPN可以正调I型干扰素的产生。2.OPN在体内也是Ⅰ型干扰素产生的强力正调者,可以诱导很强的抗病毒免疫应答。3. iOPN可以正调IRF3转录因子的活化。4. iOPN可以与重要接头分子TRAF3特异性结合。5. IOPN可以调节TRAF3的K48位偶联的泛素化修饰并且使TRAF3更稳定。6. iOPN与Triad3A竞争性结合TRAF3,进而影响Triad3A介导的,TRAF3的K48位偶联的泛素化。7. iOPN通过C端与TRAF3结合,并且其C端即可影响TRAF3的泛素化即稳定性。创新点及意义：1.本研究首次明确完善地证明iOPN可以参与调控抗病毒天然免疫反应,可以正向调节IFNβ的产生以及具有明显的抗病毒功能。这一功能的机制是由于iOPN通过抑制Triad3A与TRAF3的结合,从而抑制了Triad3A对TRAF3的K48位泛素化修饰作用,进而导致TRAF3的稳定性增强,IFNβ的活化进而增强。2.本研究提出iOPN可以抑制某分子的泛素化作用这一机制在国际上尚属少见,极大丰富了我们对于OPN功能的理解。并且我们首次发现iOPN的C端可以发挥几乎全部的抗病毒功能。3.本研究为正向调控抗病毒免疫信号通路提供了新的实验证据,为抗病毒研究以及新药物靶点的研究提供了新的思路。更多还原

【Abstract】 The innate immunity is the first line of defense against invading pathogens, which functions to respond to infection directly and relays signals for the activation of the adaptive immunity. During viral infection, multiple signaling pathways in the innate immune system are triggered to promote the production of cytokines to suppress viral replication. Central to the host antiviral response is the production of type I interferons (IFNs), which include IFN-a and IFN-(3. Several classes of termline-encoded pattern-recognition receptors (PRRs) have been linked to the production of type Ⅰ interferons during viral infection. These PRRs include Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I) like receptor (RLRs) and intracellular DNA sensors. Type I IFNs interact with its receptors then activate the JAK-STAT signal transduction pathways, leading to the induction of multiple downstream IFN-stimulated genes (ISGs).Osteopontin (OPN), also known as early T lymphocyte activation 1 (Eta-1), is a secreted glycoprotein which regulates diverse biological processes including differentiation, adhesion, bone remodeling, malignancy and immune response. OPN is considered to be a potential pro-inflammatory cytokines associated with the promotion of macrophages to secrete IFN-y and IL-12 for a long time. In recent years, with the discovery of intracellular form OPN (iOPN), different function of OPN in diverse immune cells and different stages of immune response gradually attracted people’s attention. But in innate immunity system, especially innate antiviral response, the function of iOPN still remains elusive.Objectives:Investigating the function of OPN, especially iOPN, in regulating the production of type I interferons upon viral infection. Furthermore, finding out the underlying mechanisms.Methods and Results:1. OPN expression is induced upon virus infection, and OPN especially iOPN, positively regulates IFN-β production.The expression of OPN protein and mRNA was increased after infection with SeV. Similarly, infection of VSV also increased OPN protein and mRNA expression in peritoneal macrophages. ELISA analysis showed that sOPN was also increased upon virus infection. These data demonstrated that OPN expression is induced by virus infection in murine peritoneal macrophages. To investigate the function of OPN in innate antiviral immune response, peritoneal macrophages were prepared from WT and OPN-deficient (Spp1-/-) mice and infected with SeV for various times. Then, the expression of IFN-(3 was measured. SeV infection induced the expression of IFN-P mRNA in WT macrophages. However, SeV-induced IFN-P mRNA expression was greatly decreased in OPN-deficient macrophages compared to that in WT macrophages. Consistently, OPN-deficient macrophages secreted less IFN-(3 protein than WT macrophages after SeV infection. The expression of CXCL10, Mx1 and CCL5, which are downstream genes of IFN-β signaling pathway, also decreased in SeV-infected OPN-deficient macrophages. We further investigated the function of OPN on IFN-β expression using overexpression experiments. Transfection of iOPN and full length OPN expression plasmids into HEK293 cells increased SeV-and VSV-induced IFN-β expression. Similar to IFN-β mRNA expression, transfection of iOPN and full length OPN expression plasmids also increased SeV-induced IFN-β promoter activation in a dose-dependent manner. Further, we found that addition of OPN antibody into the culture medium could not inhibit full length OPN transfection-mediated IFN-(3 activation induced by SeV infection. Overexpression of iOPN also increased RIG-I-, melanoma differentiation-associated gene 5 (MDA5)-, TRIF- and cGAS+STING-induced IFN-β promoter activation in a dose-dependent manner. In all circumstances, intracellular OPN seemed more potent to induce IFN-β expression than the full length OPN. Taken together, these data indicated that OPN, especially iOPN, positively regulates IFN-β production downstream of various innate immune signaling pathways including TLR3/4, RLRs and intracellular DNA receptor signaling2 OPN is an important positive regulator of IFN-β production and antiviral immune responses in vivo.IFN-β plays an essential role in antiviral immune response. To investigate the role of OPN in antiviral response, VSV was used to infect cells. Plaque assays showed that VSV replication greatly increased in peritoneal macrophages prepared from OPN-deficient mice compared to that from WT mice in the presence or absence of poly(l:C). Consistently, VSV RNA was also increased in OPN-deficient macrophages compared to that in WT macrophages. In contrast, overexpression of iOPN and the full length OPN in HEK293 cells greatly attenuated VSV replication in the presence or absence of poly(l:C). Taken together, these data indicated that OPN positively regulates antiviral immune response. To investigate the physiological role of OPN in antiviral response in vivo, Spp1-/- mice and WT mice were infected with VSV, and the antiviral immune responses were examined. The amount of IFN-β protein induced by VSV infection was much less in sera of VSV-infected Spp1-/- mice than that of WT mice. In accordance with reduced IFN-β production, VSV replication in the livers, spleens, and lungs was much higher in OPN-deficient (Spp1-/-) mice than in WT controls. Importantly, Spp1-/-mice were more susceptible to VSV infection than WT mice. Spp1-/- mice all died, while 50% of WT mice were alive 5 days after infection. These data suggested that OPN is an important oositive regulator of IFN-β production and antiviral immune responses.3 iOPN positively regulates IRF3 activation to regulate IFN-β production and antiviral response.IRF3 is the main transcription factor responsible for IFN-β transcription during the early phase of viral infection. To investigate the function of OPN on IRF3 activation, series of experiments were performed. First, IFN-β PRD Ⅰ/Ⅲ reporter, which harbors only IRF3 binding site in IFN-β promoter, was used. RIG-I, TRIF-and cGAS+STING-induced-IFN-p PRD Ⅰ/Ⅲ activation was increased by iOPN overexpression in a dose-dependent manner. IRF3 activation requires the phosphorylation of conserved serine and theronine residues at the c-terminal region. SeV infection induced IRF3 phosphorylation in macrophages from WT mice. While, SeV-induced IRF3 phophorylation was greatly decreased in macrophages from OPN-deficient (Spp1-/-) mice. Similarly, VSV infection-induced IRF3 phosphorylation was also greatly decreased in macrophages from Spp1-/-mice compared to that from WT mice. In contrast, overexpression of iOPN in HEK293 cells substantially increased SeV-and VSV-induced IRF3 phosphorylation. After phosphorylation, IRF3 dimerizes and translocates into nuleus to initiate IFN-(3 transcription. IRF3 dimerization was greatly decreased in OPN-deficient (Spp1-/-) macrophages compared to that in WT macrophages after SeV infection. Western blot analysis of cytoplasmic fraction and nuclear fraction showed that more IRF3 was transfected into nucleus in macrophages from WT mice compared to that from OPN-deficient (Spp1-/-) mice after virus infection. All together, these data demonstrated that OPN positively regulates IRF3 activation to regulate IFN-β production and antiviral response.4. iOPN interacts with TRAF3 to positively regulate IFN-β production and antiviral response.OPN positively regulates IFN-P production downstream of TLR3/4, RLR and intracellular DNA receptor signaling pathways, indicating OPN targets common molecules in these signaling pathways. To identify the molecules, IFN-(3 promoter activation induced by various molecules in RLRs signaling pathway was investigated. We found iOPN increased RIG-I-, MDA5-, MAVS-induced IFN-β activation, but not TBK1-and IRF3 5D-induced IFN-β activation. RT-PCR analysis of IFN-β mRNA also confirmed iOPN increased RIG-I-, MDA5-, MAVS-induced IFN-β expression, but not TBK1-and IRF3-5D-induced IFN-β expression. These data indicated that iOPN targets molecules upstream of TBK1 to positively regulate innate signaling. To directly identify iOPN targets, immunoprecipitation (IP) and western blotting (WB) were performed in HEK293 cells transfected with expression plasmids for RIG-I, MAVS, TRAF3, TNF receptor-associated factor 6 (TRAF6), STING, TBK1 and IRF3 together with iOPN. iOPN was shown to interact with TRAF3, but not with RIG-I, MAVS, STING, TBK1 and IRF3. TRAF3 has been shown to be required for the IFN-β expression downstream of TLR3/4 and RLR signaling. Interestingly, iOPN could not interact with TRAF3 homologue TRAF6, which has been shown to activate NF-κB, leading to the production of proinflammatory cytokines. Interaction between endogenous TRAF3 and OPN also detected in macrophages after SeV infection. To confirm iOPN interacts with TRAF3 directly, iOPN and TRAF3 were expressed in an in vitro protein expression system, then mixed together and followed by pull-down assays with anti-OPN antibody. TRAF3 could coimunoprecipitate with OPN, indicating a direct interaction between iOPN and TRAF3. The interactions were further supported by the colocalization studies. iOPN-GFP was found to diffuse in the cytoplasm and nucleus without SeV infection. TRAF3 was present in the cytoplasm exclusively. iOPN-GFP and TRAF3 showed less or no colocalization without SeV infection. SeV infection induced translocation of large amount of iOPN from nucleus into cytoplasm, where colocalization between iOPN and TRAF3 was greatly increased. Taken together, these data suggested that iOPN interacts with TRAF3 to positively regulate IFN-β production and antiviral response.5 iOPN inhibits K48-linked polyubiquitination and degradation of TRAF3.TRAF3 activation is tightly regulated by protein ubiquitination. K63-linked TRAF3 polyubiquitination is responsible for the activation of downstream signaling. While, K48-linked ubiquitination leads to the degradation of TRAF3 and deactivation of TRAF3-mediated downstream signaling. To investigate the molecular mechanism of iOPN in the regulation of IFN-P production, TRAF3 polyubiquitination was investigated. TRAF3 was transfected into HEK293 cells together with WT HA-ubiquitin plasmid and iOPN expression plasmid. IP and WB showed that TRAF3 polyubiquitination was greatly inhibited by iOPN expression To investigate which form of TRAF3 polyubiquitination was affected by iOPN, HA-ubiquitin mutants K48 and K63, which has only one lysine residue at position 48 and 63 respectively, were transfected into HEK293 cells together with iOPN expression plasmid. Overexpression of iOPN decreased TRAF3 polyubiquitination in HA-K48-transfected cells, whereas, TRAF3 polyubiquitination was not affected by iOPN in HA-K63-transfected cells, indicating iOPN mainly prevents TRAF3 from K48-linked polyubiquitination. K48-linked polyubiquitination leads to protein degradation by 26S proteasome. To investigate the function of iOPN on TRAF3 degradation, Myc-TRAF3 was transfected into HEK293 cells together with iOPN expression plasmid or control plasmid. After SeV infection, the half life of Myc-TRAF3 protein was measured. SeV infection led to the degradation of Myc-TRAF3 in control vector transfected cells with a half life of ～0.8 h. However, the degradation of Myc-TRAF3 was greatly attenuated in iOPN expression vector transfected cells. Similarly, SeV infection induced the degradation of Myc-TRAF3 in A549 cells, whereas, overexpression of iOPN reversed Myc-TRAF3 protein degradation after SeV infection, indicating inhibition of TRAF3 degradation by iOPN is not cell specific. To further confirm OPN stabilizes TRAF3 protein through inhibition of K48-linked ubiquitination in physiological conditions, peritoneal macrophages from WT and Spp1-/-mice were prepared and infected with SeV. IP and WB showed that K48-linked polyubiquitination of TRAF3 was greatly increased in the macrophages from Spp1-/-mice compared to that in macrophages from WT mice after SeV infection. Increased K48-linked TRAF3 polyubiquitination in OPN-deficient macrophages was further confirmed with proteins immunoprecipitated with anti-TRAF3 under stringent conditions. Consistent with more TRAF3 ubiquitination, TRAF3 was degraded more rapidly in OPN-deficient macrophages. All together, these data indicated that OPN prevents TRAF3 from K48-linked polyubiquitination and degradation.6. iOPN inhibits Triad3A-mediated TRAF3 polyubiquitination.iOPN alone does not have the ability to modulate protein ubiquitination. There are two possibilities for iOPN to inhibit TRAF3 polyubiquitination. One is that iOPN may recruit deubiquitinating enzymes (DUB) to cleave K48-linked polyubiquitin chains from TRAF3 to stabilize TRAF3. Recently, USP25 has been reported to cleave K48-linked polyubiquitin chains from TRAF3. To investigate whether iOPN recruit USP25 to stabilize TRAF3, TRAF3 ubiquitination was measured in the presence of USP25 and iOPN expression plasmids. iOPN expression decreased TRAF3 ubiquitination. USP25 expression indeed decreased TRAF3 polyubiquitination. While, cotransfection of USP25 and iOPN could not further decrease USP25-mediated deubiquitination from TRAF3, suggesting iOPN may not recruit USP25 to cleave K48-linked ubiquitin from TRAF3. Another possibility is that iOPN prevents an E3 ligase from binding to TRAF3. Triad3A has been reported to be an E3 ligase involved in TRAF3 ubiquitination and degradation after virus infection. To investigate whether iOPN inhibits Triad3A-mediated TRAF3 ubiquitination, TRAF3 was transfected into HEK293 cells together with Triad3A and iOPN. IP and WB showed that Triad3A promoted TRAF3 ubiquitination. Overexpression of iOPN greatly decreased TRAF3 ubiquitination mediated by Triad3A. In vitro ubiquitination assays with in vitro expressed proteins also confirmed that Triad3A-induced K48-linked TRAF3 polyubiqutination was greatly attenuated by iOPN. Triad3A binding to TRAF3 was also decreased by iOPN in a dose-dependent manner. In vitro pull-down assays confirmed the binding Triad3A to TRAF3 was gradually decreased with the increasing binding of iOPN to TRAF3. Consistent with the inhibition of Triad3A-induced TRAF3 ubiqutination, Triad3A-induced degradation of TRAF3 was reversed by iOPN expression. The Y residue and Q residue at position 441 and 443 of TRAF3 have been reported for Triad3A binding. To confirm the importance of these two residues, TRAF3 mutant was constructed by mutating Y441 and Q443 to A. Mutation of YQ to AA ablated Triad3A binding to TRAF3. Notably, iOPN binding to TRAF3 mutant was also ablated, indicating iOPN binding to the same sites in TRAF3 as the Triad3A. All together, these data demonstrated that iOPN competes with Trida3A for the binding to TRAF3, which prevents TRAF3 from K48-linked polyubiquitination and degradation promoted by Triad3A.7. C-terminal fragment of iOPN binds to TRAF3.Endogenous OPN can be cleaved by thrombin at position 168 into two fragments. In order to investigate the OPN fragment involved in the binding and regulation of TRAF3 ubiquitination, two OPN truncations were constructed and expressed in vitro. In vitro pull-down assays demonstrated that full length and the C-terminal fragment of iOPN, but not the N-terminal fragment, bound to TRAF3. Consistent with the C-terminal fragment binding to TRAF3, Trida3A-induced TRAF3 ubiqutination was inhibited by the C-terminal fragment, but not the N-terminal fragment. These data indicated that the C-terminal fragment of OPN is responsible for the binding and inhibition of ubiquitination of TRAF3. To investigate the inhibition of TRAF3 ubiquitination by WT and the C-terminal fragment of iOPN has a physiological role on IFN-P production, lentiviral expression plasmids for WT, N-terminal fragment and C-terminal fragment of iOPN were constructed and used to infect WT and OPN-deficient macrophages. Infection of lentivirus containing WT iOPN plasmid increased OPN expression in WT macrophages and restored iOPN expression in OPN-deficient (Spp1-/-) macrophages. Consistent with positive function of iOPN on IFN-(3 production, lentiviral infection of WT iOPN expression plasmid into WT macrophages further increased SeV-induced expression of IFN-β, CXCL10, Mx1 and CCL5. Lentiviral infection of WT iOPN expression plasmid into OPN-deficient macrophages restored SeV-induced expression of IFN-β, CXCL10, Mx1 and CCL5 to the same level as that in WT macrophages. Consistent with ability to inhibit TRAF3 ubiquitination by the C-terminal fragment, lentiviral infection of the C-terminal fragment of iOPN increased SeV-induced expression of IFN-(3, CXCL10, Mx1 and CCL5 in WT macrophages. SeV-induced expression of IFN-β in OPN-deficient macrophages was also restored upon infection with lentivirus containing the C-terminal fragment of iOPN. But, infection of lentivirus containing the N-terminal fragment of iOPN could not increase or restore SeV-induced expression of IFN-βin WT and OPN-deficient macrophages, respectively Taken together, these data demonstrated that iOPN binds to TRAF3 through the C-terminal fragment, preventing TRAF3 from K48-linked ubiquitination and degradation and leading to increased IFN-β production and innate antiviral response.Conclusions:1. OPN expression is induced upon virus infection, and OPN especially iOPN, positively regulates IFN-β production.2. OPN is an important positive regulator of IFN-(3 production and antiviral immune responses in vivo.3. iOPN positively regulates IRF3 activation to regulate IFN-β production and antiviral response.4. iOPN interacts with TRAF3 to positively regulate IFN-(3 production and antiviral response.5. iOPN inhibits K48-linked polyubiquitination and degradation of TRAF3.6. iOPN compete with Trida3A for the binding to TRAF3, which prevents TRAF3 from K48-linked polyubiquitination and degradation promoted by Triad3A.7. iOPN binds to TRAF3 through the C-terminal fragment, preventing TRAF3 from K48-linked ubiquitination and degradation and leading to increased IFN-β production and innate antiviral response.Innovation and significance:1. To the best of our knowledge, this study is the first to completely prove that iOPN can positively regulate IFN-β production and antiviral innate immune response.2. We first time illustrated that the C terminal of iOPN could inhibit the ubiquitination of any protein through competitive combination.3. These researches improve the function of iOPN in innate immunity, propose new understanding about expression regulation mechanism of type I interferons, and provide the theoretical basis for the relevant drug development.更多还原