鸡TIA-1和G3BP1在应激颗粒形成和抗病毒反应过程中的作用

发布时间：2018-12-13 04:40

【摘要】：当细胞暴露于各种环境应激条件下,如亚砷酸盐,缺氧和热休克时,细胞终止蛋白翻译,在细胞质中形成储存mRNA的颗粒,称为应激颗粒(stress granule,SG)。SG是在应激条件下从解聚的多核糖体储存位点释放的mRNA,对选择性翻译是所必需的。当应激条件消失时,应激刺激的细胞内损伤得到修复,例如DNA损伤和错误折叠蛋白的积累。RNA结合蛋白T细胞内部抗原-1 (TIA-1)和GTP酶激活蛋白SH3功能区结合蛋白1 (G3BP1)是参与SG形成的重要组件。作为对环境应激的一种保守应答反应,SG存在于各种物种中。然而,在鸡细胞中SG形成以及TIA-1/G3BP1在鸡SG装配中的作用还没有被阐明。本研究重点将对鸡TIA-1/G3BP1的功能及其在病毒复制中的作用进行研究。TIA-1在调节人类细胞中前mRNA剪接,mRNA翻译和SG形成中发挥作用。在本研究中,我们克隆鸡TIA-1 (cTIA-1),结果表明外源转染cTIA-1促进人和鸡细胞中经典SGs的形成。具有氨基末端GFP标签(ntGFP-cPRD)或Flag标签(Flag-cPRD)的cTIA-1朊蛋白相关结构域cPRD的过表达诱导典型的SG的产生,说明cPRD参与了SG的聚集。然而,羧基末端GFP标签(ctGFP-cPRD) cPRD诱导明显较大的细胞质颗粒,并且不与内源性G3BP1共定位,CHX处理后仍保持稳定不降解,这些结果表明这些颗粒不是典型的SGs,ctGFP标签影响cPRD定位。在外界环境应激下,内源性cTIA-1被募集到的鸡细胞的SG中,在热休克处理下,鸡心脏中出现明显SG颗粒。以上结果表明:cTIA-1在鸡细胞中的经典SG形成中起作用,cPRD在SG聚集过程中发挥重要作用。G3BP1是SGs的重要组件,能够启动组装SGs形成多聚体。为掌握G3BP1在如何作用于鸡细胞中SG形成,以及鸡G3BP1 (cG3BP1)在新城疫病毒(NDV)感染诱导SG形成过程中的作用,用NDV感染HeLa细胞,免疫荧光检测内源性G3BP1的定位,结果表明G3BP1呈现典型的点状荧光,但不与病毒P蛋白共定位。将鸡G3BP1分别转染至HeLa, DF-1和CEF后,以不同应激处理之后均能在细胞中观察到SG。外源转染cG3BP1和各分段结构域后以不同应激处理之后观察与内源性SG标志物共定位现象,结果发现G3BP1的A结构域(NTF2样结构域)不能被招募到SGs中,并且抑制原有SGs的形成,D区为RNA结合结构域,单独表达D区能够诱导SG形成,说明cG3BP1的RNA结合结构域对聚集至关重要。最后转染G3BP1之后感染NDV,以Western-blot和TCID50检测病毒复制情况。结果显示:G3BP1过表达能够增强病毒细胞中病毒蛋白的表达以及上清中的病毒滴度,说明SG在NDV复制过程中发挥促进作用。鸡TIA-1和G3BP1在鸡细胞SG形成过程中至关重要,鸡细胞中G3BP1介导的SG能够促进NDV复制,这为进一步研究NDV和禽源细胞互作奠定了基础。
[Abstract]:When cells are exposed to various environmental stresses, such as arsenite, anoxia and heat shock, cell termination proteins translate into particles that store mRNA in the cytoplasm, known as stress particles (stress granule, SG). SG is the release of mRNA, from depolymerized polyribosomal storage sites under stress conditions, which is necessary for selective translation. When the stress condition disappeared, the stress-stimulated intracellular damage was repaired. For example, DNA damage and the accumulation of misfolded proteins. RNA binding protein T cell internal antigen 1 (TIA-1) and GTP enzyme activating protein SH3 binding protein 1 (G3BP1) are important components involved in the formation of SG. As a conservative response to environmental stress, SG exists in a variety of species. However, the formation of SG in chicken cells and the role of TIA-1/G3BP1 in chicken SG assembly have not been clarified. This study focuses on the function of chicken TIA-1/G3BP1 and its role in viral replication. TIA-1 plays a role in regulating premRNA splicing, mRNA translation and SG formation in human cells. In this study, we cloned chicken TIA-1 (cTIA-1). The results showed that exogenous transfection of cTIA-1 promoted the formation of classical SGs in human and chicken cells. The overexpression of cTIA-1 prion protein related domain cPRD with amino terminal GFP tag (ntGFP-cPRD) or Flag tag (Flag-cPRD) induces the production of typical SG, indicating that cPRD is involved in the aggregation of SG. However, the carboxyl terminal GFP label (ctGFP-cPRD) cPRD induced significantly larger cytoplasmic particles and did not co-locate with endogenous G3BP1, and CHX remained stable and non-degradable after treatment with CHX. These results indicated that these particles were not typical SGs,. CtGFP tags affect cPRD positioning. In the SG of chicken cells raised by endogenous cTIA-1 under external environmental stress, obvious SG granules appeared in chicken heart under heat shock treatment. These results indicate that cTIA-1 plays an important role in the formation of classical SG in chicken cells and cPRD plays an important role in the aggregation of SG. G3BP1 is an important component of SGs and can initiate the assembly of SGs to form a polymer. In order to understand the role of G3BP1 in the formation of SG in chicken cells and the role of G3BP1 (cG3BP1) in SG formation induced by Newcastle disease virus (NDV) infection, HeLa cells were infected with NDV and the localization of endogenous G3BP1 was detected by immunofluorescence. The results showed that G3BP1 showed typical dot fluorescence, but not co-located with virus P protein. After transfection of chicken G3BP1 into HeLa, DF-1 and CEF, SG. could be observed in the cells after different stress treatments. After exogenous transfection of cG3BP1 and each segmental domain, the co-localization with endogenous SG markers was observed after different stress treatments. The results showed that the A domain (NTF2 like domain) of G3BP1 could not be recruited into SGs and inhibited the formation of original SGs. The expression of D region alone can induce the formation of SG, indicating that the RNA binding domain of cG3BP1 plays an important role in aggregation. Finally, after transfection of G3BP1, NDV, was infected with Western-blot and TCID50 to detect the replication of the virus. The results showed that the overexpression of G3BP1 could enhance the expression of viral protein and the titer of virus in the supernatant, indicating that SG played a promoting role in the process of NDV replication. Chicken TIA-1 and G3BP1 play an important role in the formation of chicken cell SG. SG mediated by G3BP1 in chicken cells can promote NDV replication, which lays a foundation for further study on the interaction between NDV and avian origin cells.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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