SFTSV NSs抑制IFN-β產(chǎn)生機(jī)制的研究
發(fā)布時(shí)間：2018-10-21 17:47
【摘要】：發(fā)熱伴血小板減少綜合征病毒(Severe fever with thrombocytopenia syndrome virus, SFTSV)為我國(guó)2010年發(fā)現(xiàn)的一種布尼亞病毒科白蛉病毒屬的新病毒,是發(fā)熱伴血小板減少綜合征(SFTS)的病原體,SFTS的主要臨床表現(xiàn)為發(fā)熱、血小板減少,病情嚴(yán)重者可引發(fā)多臟器損傷甚至死亡,平均病死率高達(dá)10%。SFTSV基因組為分節(jié)段的單股負(fù)鏈RNA,分為L(zhǎng)、M和S三個(gè)基因片段,其中S片段具有兩個(gè)方向相反的閱讀框,可以編碼核蛋白(NP)和非結(jié)構(gòu)蛋白(NSs)。非結(jié)構(gòu)蛋白并不是病毒顆粒的組成部分,而是主要存在于感染的宿主細(xì)胞中,目前我們對(duì)NSs在宿主體內(nèi)的具體功能和臨床意義尚不清楚。病毒若要對(duì)機(jī)體產(chǎn)生有效感染,就必須成功逃脫天然免疫系統(tǒng)的攻擊,同時(shí),SFTSV患者體內(nèi)的干擾素含量非常低,提示SFTSV具有逃逸天然免疫系統(tǒng)的功能。但是其具體的天然免疫逃逸機(jī)制目前還知之甚少,因此本文擬從天然免疫方面入手來研究SFTSV的天然免疫逃逸機(jī)制。 天然免疫系統(tǒng)是機(jī)體抵抗病毒入侵的第一道防線,其中干擾素又是抗病毒的最重要分子。干擾素-p是由纖維細(xì)胞產(chǎn)生,而纖維細(xì)胞同時(shí)又是許多病毒入侵的重要細(xì)胞之一。因此,我們首先通過報(bào)告基因來確定SFTSV對(duì)IFN-β表達(dá)的影響,然后尋找SFTSV影響IFN-β表達(dá)的具體蛋白,在此基礎(chǔ)上確定病毒蛋白(?)響IFN-p表達(dá)的作用分子及與該分子的作用位點(diǎn)。實(shí)驗(yàn)結(jié)果證實(shí)了SFTSV可以抑制機(jī)體IFN-p的表達(dá),并且確定SFTSV的非結(jié)構(gòu)蛋白(NSs)對(duì)IFN-β的表達(dá)存在較強(qiáng)的抑制作用。隨后我們通過免疫共沉淀、質(zhì)譜檢測(cè)和Western blot等試驗(yàn)研究了SFTSV非結(jié)構(gòu)蛋白(NSs)抑制機(jī)體IFN-p表達(dá)的具體機(jī)制：非結(jié)構(gòu)蛋白與細(xì)胞IFN-p形成通路中的TBK1分子發(fā)生特異性結(jié)合,這種結(jié)合對(duì)宿主TBK1的表達(dá)和磷酸化無明顯的影響,但是NSs與TBK1的結(jié)合作用可以抑制下游IRF3分子的磷酸化和二聚體的形成,從而抑制IFN-p的產(chǎn)生,以便SFTSV成功逃逸宿主細(xì)胞天然免疫系統(tǒng)的攻擊,進(jìn)而造成有效感染。在此基礎(chǔ)上,我們對(duì)SFTSVNSs的功能位點(diǎn)進(jìn)行了研究,結(jié)果顯示NSs抑制機(jī)體IFN-p表達(dá)的功能位點(diǎn)主要位于N端的25位氨基酸附近,將其缺失后NSs對(duì)IFN-β的抑制作用會(huì)明顯降低,與TBK1的結(jié)合能力也會(huì)明顯減弱。綜上所述,SFTSV NSs蛋白通過與IFN-β產(chǎn)生的信號(hào)通路中的TBK1分子發(fā)生特異性結(jié)合而阻斷通路下游分子的活化,進(jìn)而抑制IFN-p的產(chǎn)生,并且NSs的主要作用位點(diǎn)為N端的25位附近的氨基酸。本研究為SFTSV的防治工作提供了理論指導(dǎo)和技術(shù)支持,并為病毒的免疫機(jī)制研究作出一些貢獻(xiàn)。
[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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R511
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R511
【共引文獻(xiàn)】
相關(guān)期刊論文 前1條
1 韓翡;焦培榮;韋良孟;何靜;宋亞芬;康銀峰;崔進(jìn);張爍;廖明;任濤;;鴿TLR7基因的克隆、鑒定及表達(dá)分析[J];華南農(nóng)業(yè)大學(xué)學(xué)報(bào);2014年03期
相關(guān)博士學(xué)位論文 前1條
1 王蓉;豬繁殖與呼吸綜合征病毒對(duì)Ⅰ型干擾素下游信號(hào)通路影響的研究[D];西北農(nóng)林科技大學(xué);2013年
，本文編號(hào)：2285881
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