SFTSV NSs抑制IFN-β产生机制的研究
发布时间：2018-10-21 17:47
【摘要】：发热伴血小板减少综合征病毒(Severe fever with thrombocytopenia syndrome virus, SFTSV)为我国2010年发现的一种布尼亚病毒科白蛉病毒属的新病毒,是发热伴血小板减少综合征(SFTS)的病原体,SFTS的主要临床表现为发热、血小板减少,病情严重者可引发多脏器损伤甚至死亡,平均病死率高达10%。SFTSV基因组为分节段的单股负链RNA,分为L、M和S三个基因片段,其中S片段具有两个方向相反的阅读框,可以编码核蛋白(NP)和非结构蛋白(NSs)。非结构蛋白并不是病毒颗粒的组成部分,而是主要存在于感染的宿主细胞中,目前我们对NSs在宿主体内的具体功能和临床意义尚不清楚。病毒若要对机体产生有效感染,就必须成功逃脱天然免疫系统的攻击,同时,SFTSV患者体内的干扰素含量非常低,提示SFTSV具有逃逸天然免疫系统的功能。但是其具体的天然免疫逃逸机制目前还知之甚少,因此本文拟从天然免疫方面入手来研究SFTSV的天然免疫逃逸机制。 天然免疫系统是机体抵抗病毒入侵的第一道防线,其中干扰素又是抗病毒的最重要分子。干扰素-p是由纤维细胞产生,而纤维细胞同时又是许多病毒入侵的重要细胞之一。因此,我们首先通过报告基因来确定SFTSV对IFN-β表达的影响,然后寻找SFTSV影响IFN-β表达的具体蛋白,在此基础上确定病毒蛋白(?)响IFN-p表达的作用分子及与该分子的作用位点。实验结果证实了SFTSV可以抑制机体IFN-p的表达,并且确定SFTSV的非结构蛋白(NSs)对IFN-β的表达存在较强的抑制作用。随后我们通过免疫共沉淀、质谱检测和Western blot等试验研究了SFTSV非结构蛋白(NSs)抑制机体IFN-p表达的具体机制：非结构蛋白与细胞IFN-p形成通路中的TBK1分子发生特异性结合,这种结合对宿主TBK1的表达和磷酸化无明显的影响,但是NSs与TBK1的结合作用可以抑制下游IRF3分子的磷酸化和二聚体的形成,从而抑制IFN-p的产生,以便SFTSV成功逃逸宿主细胞天然免疫系统的攻击,进而造成有效感染。在此基础上,我们对SFTSVNSs的功能位点进行了研究,结果显示NSs抑制机体IFN-p表达的功能位点主要位于N端的25位氨基酸附近,将其缺失后NSs对IFN-β的抑制作用会明显降低,与TBK1的结合能力也会明显减弱。综上所述,SFTSV NSs蛋白通过与IFN-β产生的信号通路中的TBK1分子发生特异性结合而阻断通路下游分子的活化,进而抑制IFN-p的产生,并且NSs的主要作用位点为N端的25位附近的氨基酸。本研究为SFTSV的防治工作提供了理论指导和技术支持,并为病毒的免疫机制研究作出一些贡献。
[Abstract]:Fever with thrombocytopenia syndrome virus (Severe fever with thrombocytopenia syndrome virus, SFTSV) is a new virus of the genus Phledoviridae found in China in 2010. It is the pathogen of fever with thrombocytopenia syndrome (SFTS). The main clinical manifestation of SFTS is fever. Thrombocytopenia and severe disease can lead to multiple organ injury or even death. The average death rate is as high as single strand negative strand RNA, which is segmented into segments of 10%.SFTSV genome, which is divided into three gene fragments, Lomb M and S, in which S fragment has two reading frames in opposite direction. Can encode nuclear protein (NP) and nonstructural protein (NSs). Non-structural proteins are not part of viral particles, but mainly exist in infected host cells. At present, we do not know the specific function and clinical significance of NSs in the host. If the virus is to infect the body effectively, it must successfully escape the attack of the innate immune system. At the same time, the level of interferon in the patients with SFTSV is very low, which suggests that SFTSV has the function of escaping the innate immune system. However, little is known about the mechanism of innate immune escape, so this paper intends to study the mechanism of innate immune escape of SFTSV from the aspect of innate immunity. Innate immune system is the first line of defense against virus invasion, in which interferon is the most important antiviral molecule. Interferon-p (IFN-p) is produced by fibroblasts, and fibroblasts are one of the most important cells invading by many viruses. Therefore, we first determine the effect of SFTSV on the expression of IFN- 尾 through the reporter gene, and then find the specific protein of SFTSV that affects the expression of IFN- 尾, and then determine the viral protein (?) The interacting molecule and the interaction site with IFN-p. The results showed that SFTSV could inhibit the expression of IFN-p, and the SFTSV non-structural protein (NSs) had a strong inhibitory effect on the expression of IFN- 尾. Then we studied the specific mechanism of the inhibition of IFN-p expression by SFTSV non-structural protein (NSs) through immunoprecipitation, mass spectrometry and Western blot tests: the non-structural proteins specifically bind to TBK1 molecules in the IFN-p formation pathway of cells. This binding has no significant effect on the expression and phosphorylation of host TBK1, but the binding of NSs to TBK1 can inhibit the phosphorylation of downstream IRF3 molecules and the formation of dimers, thereby inhibiting the production of IFN-p. In order to escape the attack of innate immune system of host cells successfully, SFTSV can cause effective infection. On this basis, we studied the functional sites of SFTSVNSs. The results showed that the functional sites of NSs to inhibit the expression of IFN-p were mainly located near the 25 amino acids at the N-terminal, and the inhibitory effect of NSs on IFN- 尾 was significantly decreased after the deletion of NSs. The ability to bind to TBK1 is also significantly weakened. In conclusion, SFTSV NSs protein inhibits the activation of downstream IFN-p molecules by specifically binding to TBK1 molecules in the IFN- 尾 signaling pathway, and the main site of NSs is amino acids near the N-terminal site. This study provides theoretical guidance and technical support for the prevention and treatment of SFTSV and contributes to the study of the immune mechanism of the virus.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R511
本文编号：2285881
[Abstract]:Fever with thrombocytopenia syndrome virus (Severe fever with thrombocytopenia syndrome virus, SFTSV) is a new virus of the genus Phledoviridae found in China in 2010. It is the pathogen of fever with thrombocytopenia syndrome (SFTS). The main clinical manifestation of SFTS is fever. Thrombocytopenia and severe disease can lead to multiple organ injury or even death. The average death rate is as high as single strand negative strand RNA, which is segmented into segments of 10%.SFTSV genome, which is divided into three gene fragments, Lomb M and S, in which S fragment has two reading frames in opposite direction. Can encode nuclear protein (NP) and nonstructural protein (NSs). Non-structural proteins are not part of viral particles, but mainly exist in infected host cells. At present, we do not know the specific function and clinical significance of NSs in the host. If the virus is to infect the body effectively, it must successfully escape the attack of the innate immune system. At the same time, the level of interferon in the patients with SFTSV is very low, which suggests that SFTSV has the function of escaping the innate immune system. However, little is known about the mechanism of innate immune escape, so this paper intends to study the mechanism of innate immune escape of SFTSV from the aspect of innate immunity. Innate immune system is the first line of defense against virus invasion, in which interferon is the most important antiviral molecule. Interferon-p (IFN-p) is produced by fibroblasts, and fibroblasts are one of the most important cells invading by many viruses. Therefore, we first determine the effect of SFTSV on the expression of IFN- 尾 through the reporter gene, and then find the specific protein of SFTSV that affects the expression of IFN- 尾, and then determine the viral protein (?) The interacting molecule and the interaction site with IFN-p. The results showed that SFTSV could inhibit the expression of IFN-p, and the SFTSV non-structural protein (NSs) had a strong inhibitory effect on the expression of IFN- 尾. Then we studied the specific mechanism of the inhibition of IFN-p expression by SFTSV non-structural protein (NSs) through immunoprecipitation, mass spectrometry and Western blot tests: the non-structural proteins specifically bind to TBK1 molecules in the IFN-p formation pathway of cells. This binding has no significant effect on the expression and phosphorylation of host TBK1, but the binding of NSs to TBK1 can inhibit the phosphorylation of downstream IRF3 molecules and the formation of dimers, thereby inhibiting the production of IFN-p. In order to escape the attack of innate immune system of host cells successfully, SFTSV can cause effective infection. On this basis, we studied the functional sites of SFTSVNSs. The results showed that the functional sites of NSs to inhibit the expression of IFN-p were mainly located near the 25 amino acids at the N-terminal, and the inhibitory effect of NSs on IFN- 尾 was significantly decreased after the deletion of NSs. The ability to bind to TBK1 is also significantly weakened. In conclusion, SFTSV NSs protein inhibits the activation of downstream IFN-p molecules by specifically binding to TBK1 molecules in the IFN- 尾 signaling pathway, and the main site of NSs is amino acids near the N-terminal site. This study provides theoretical guidance and technical support for the prevention and treatment of SFTSV and contributes to the study of the immune mechanism of the virus.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R511
【共引文献】
相关期刊论文 前1条
1 韩翡;焦培荣;韦良孟;何静;宋亚芬;康银峰;崔进;张烁;廖明;任涛;;鸽TLR7基因的克隆、鉴定及表达分析[J];华南农业大学学报;2014年03期
相关博士学位论文 前1条
1 王蓉;猪繁殖与呼吸综合征病毒对Ⅰ型干扰素下游信号通路影响的研究[D];西北农林科技大学;2013年
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