摘要
口蹄疫(Foot-and-Mouth Disease, FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus, FMDV)引起的一种急性、热性、高度接触传染性动物疫病,是全球范围内家畜及其产品贸易最大的羁绊。FMDV通过逃避宿主的免疫监视建立持续性感染,使患畜持续向外界排毒,成为传染源。VP1是FMDV主要的结构蛋白,可参与病毒入侵和免疫逃逸,但VP1在细胞中发挥作用的机理尚不清楚。本研究通过寻找与VP1相互作用的宿主靶蛋白,初步阐明VP1在FMDV引起的免疫抑制中所发挥的作用。
本研究通过双荧光素酶报告基因检测法和荧光定量PCR发现FMDV VP1显著抑制Ⅰ型干扰素和NF-κB的活化。因此,推测VP1在FMDV抑制宿主细胞Ⅰ型干扰素表达的过程中起着关键作用。为了探索FMDV VP1抑制Ⅰ型干扰素产生的分子机理,应用酵母双杂交技术寻找与FMDVVP1相互作用的宿主细胞靶蛋白,结果显示VP1与可溶性耐药相关钙结合蛋白(soluble resistance-related calcium-binding protein, Sorcin)存在着相互作用,并通过免疫共沉淀和激光共聚焦技术验证了两者在细胞中的相互作用。之后,利用RNAi沉默细胞内源性Sorcin蛋白的表达,发现Sorcin是VP1抑制细胞Ⅰ型干扰素产生所必需的蛋白。进一步研究发现：Sorcin会影响细胞中Ⅰ型干扰素和NF-κB的转录激活水平,表明Sorcin是免疫信号传导通路中的一个负调控分子。此外,构建了Sorcin基因敲除小鼠,以期在体内验证Sorcin的作用,初步的实验结果表明：基因敲除小鼠较野生型小鼠表现出更为明显的炎症反应。
为了揭示Sorcin负调控的机制,本文利用酵母双杂交技术,以Sorcin蛋白为诱饵,筛选人白细胞文库中与其相互作用的蛋白,找到新的靶蛋白信号传导和转录激活因子-3(signal transducer and activator of transcription3, STAT3),并通过免疫共沉淀和激光共聚焦实验验证两者的相互作用,证明STAT3是Sorcin在免疫信号传递通路中下游的接头分子。
综上所述,本研究发现在FMDV感染宿主细胞的过程中,VP1蛋白通过与宿主细胞蛋白Sorcin的互作,进而活化转录因子STAT3, STAT3参与负调控NF-κB信号通路,最终抑制宿主细胞Ⅰ型干扰素的产生,为深入了解FMDV造成持续感染的分子机理提供了重要参考。
Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious animal disease caused by FMD virus (FMDV), and has been recognized as the most important constraint to international trade in animals and animal products. An outstanding feature for FMDV infection is that the FMDV infected animals may remain as a carrier state, some of the animals exposed to FMDV may have a long term asymptomatic infection. Viruses can evade host immune surveillance and exploit the host response to provide favorable intracellular conditions for long-term persistence. FMDV VP1is the major structural protein, which plays an important role in the process of viral infection and immune evasion, but the underlying mechanism has not yet been elucidated. Here we showed the critical role of VP1in FMDV-induced persistent infection.
In this study, we found that the FMDV VP1was transfected into HEK293T cells and the effects of expression of the viral proteins on expression of type I interferon and the transcription activation of NF-κB were examined by the qRT-PCR and dual-luciferase reporter gene assay. To further investigate the molecular mechanism underlying VP1-induced immunosuppression, we performed the Yeast Two-hybrid assay using FMDV protein VP1as the bait and the PK15cDNA library as the prey to screen the VP1-interacting proteins. Among the positive clones, we focus on soluble resistance-related calcium-binding protein (Sorcin). The interaction between VP1and Sorcin was confirmed by Co-IP and Confocal assay. Importantly, we found that knockdown of Sorcin abolished the inhibitory effects of the activation of Type I interferon and NF-κB promoters. These results indicate that Sorcin is required for VP1-induced suppression of type I interferon response. In addition, over-expression or down-expression of Sorcin can also affect the transcriptional activation of type I interferons and NF-κB, it is speculated that the Sorcin is a negative regulator of immune signaling pathway. To further validate the role of Sorcin in vivo, constructing Sorcin knockout mice, preliminary experimental results showed that: knockout mice exhibited more severe inflammation.
To explore the mechanism of action of Sorcin and the pathways in which Sorcin is involved, yeast two hybrid screens, co-immunoprecipitation and confocal assay unambiguously identified signal transducer and activator of transcription3(STAT3) as a Sorcin-interacting protein. More interesting, knocking down of the STAT3abolished the ability of Sorcin to inhibit NF-κB signaling, suggesting that STAT3functions downstream of Sorcin in NF-κB signaling pathway.
In summary, by yeast two-hybrid screening and immunoprecipitation approaches, we found that FMDV VP1interacted with Sorcin in the cytoplasm, and that VP1inhibited type I IFN response via interacting with Sorcin. Importantly, we found that Sorcin interacts with STAT3, and that STAT3act as an downstream adaptor protein of Sorcin in the negative regulation of NF-κB signaling pathway. These data further our understandings and provide new insights into the molecular mechanisms of the persistent infection of FMDV.
引文
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