轮状病毒编码小RNA分子1755激活细胞自噬及调控自身复制的机制

发布时间：2018-04-14 06:16

本文选题：轮状病毒 + microRNAs　；参考：《北京协和医学院》2017年博士论文

【摘要】：宿主细胞与病毒的相互作用研究对揭示病毒的复制机制具有重要意义。在病毒感染过程中,病毒能够编码microRNA分子调控自身基因及宿主基因的表达,促进病毒的复制及逃避宿主的免疫监控。研究表明,在病毒感染过程中,病毒能够劫持宿主细胞的自噬膜系统来实现病毒自身的复制。在这个过程中有多个调控环节和因素参与,但劫持的具体机制还不清楚,尚需探讨。轮状病毒(Rotavirus,RV)是一种基因组分节段的双链RNA病毒,为呼肠病毒科,轮状病毒属,在引起婴幼儿腹泻中有重要的病原学及流行病学地位。论文围绕RV复制的机制,特别是对感染宿主细胞后的细胞自噬调控机理以及对病毒复制能力的相互关系进行了深入细致的研究。这项研究不仅对揭示病毒复制的分子机制、对致病机理探讨以及药物靶点筛选均有重要意义。在这项研究中我们采用了小RNA深度测序的方法,对从流行区分离的野毒株ZTR68(G1P[8]型)感染宿主细胞MA104(猴肾传代细胞系)后所产生的差异表达miRNAs进行了筛选,发现其中小RNA分子chr-1755表达明显高于其他,因此推测该分子可能在RV复制中发挥主要作用,将其命名为vsRNA1755。为了证实该分子在不同病毒型和不同细胞中是否存在表达普遍性,实验选用了另外4个不同G型(G2、G3、G4、G9)的RV和两种细胞HT-29(人结肠癌细胞系)、Caco-2(人结直肠腺癌细胞系)进行了对比,发现vsRNA1755均有高表达。随后对G1型ZTR68毒株感染MA104细胞后的一系列调控机制做了深入研究。在病毒早期复制能力与vsRNA1755关系中发现二者呈正相关性。通过BLAST比对及体外转染NSP4基因分析表明,vsRNA1755来源于RV的非结构蛋白NSP4(编码肠毒素与病毒RNA复制相关基因)基因,是由NSP4基因coil-coiled区域编码的小RNA分子,生物合成中由Dicer-1和AGO1参与。为了寻找vsRNA1755的下游靶基因,通过miRanda软件预测,揭示胰岛素样生长因子 1 受体(Insulin-like Growth Factor 1 Receptor,IGF1R)是 vsRNA1755 可能靶点。我们用双荧光素酶报告系统,经体外转染vsRNA1755模拟物(mimics)转染细胞检测表达情况,证明二者存在靶定关系,vsRNA1755能够下调IGF1R的表达。在获得了靶基因关系后,我们对vsRNA1755的生物学功能进行了探讨,考虑到IGF1R是PI3K/Akt通路的起始元件且与自噬通路的激活密切相关,我们的实验集中在vsRNA1755与自噬发生的机理研究上。通过自噬流检测试剂盒(特异性检测)及电镜形态学检测发现,当转染了自噬标志物LC3蛋白再感染RV后,LC3-I转换为LC3-II,说明RV感染后的细胞发生了明确的自噬现象。共转染绿色荧光蛋白(EGFP)与LC3的示踪结果显示,RV与LC3存在共定位关系,进一步验证RV感染与自噬有密切关系。形态学观察发现,感染后出现自噬小体,感染4小时明显增多,后期则可见形成的病毒池变大,病毒颗粒数增加。当用自噬抑制剂3-MA后,病毒复制能力下降,提示自噬促进了病毒复制。在研究vsRNA1755与自噬调控机制实验中,我们用该分子的模拟物(mimics)转染细胞检测了多个自噬调控蛋白(ATG5、ATG12、P62)的表达,发现自噬相关蛋白ATG5和ATG12表达上调,自噬底物P62表达下调。蛋白印迹实验显示,过表达vsRNA1755后,自噬特异性底物P62蛋白明显下调。通过加入PI3K/Akt通路抑制剂LY294002,检测病毒的感染及拷贝数,结果显示,PI3K/Akt通路对病毒的复制起抑制作用。对vsRNA1755与靶基因IGF1R进行的qRT-PCR和蛋白印迹检测,结果提示,RV在感染早期可以下调IGF1R的表达,推测vsRNA1755通过下调IGF1R抑制PI3K/Akt/mTOR信号通路的活化,激活了自噬,促进病毒复制。体外转染mimics活化上调了该通路的结果也印证了这一可能性。综上所述,RV感染宿主细胞后,由RV的NSP4基因编码的小RNA分子1755(vsRNA1755),通过抑制IGF1R表达引起下游PI3K/Akt/mTOR信号通路失活,解除对细胞自噬通路的抑制,激活细胞自噬,病毒劫持了细胞的自噬通路促进了自身的复制。研究的结果不仅为揭示RV感染机制提供了主要依据,也为病毒与宿主细胞间的相互作用及未来有针对性、更加精准的设计药物靶点提供了有意义的理论依据。
[Abstract]:It is important to study the interaction between host cell and virus replication mechanism to reveal the virus. During viral infection, the virus can express microRNA encoding gene and its molecular regulation of host genes to promote viral replication and evade the host immune surveillance. The results show that during viral infection, the virus can hijack host cell the autophagic membrane system to achieve viral replication. In many regulatory factors in this process, but the specific mechanism of hijacking is not clear, still need further discussion. Rotavirus (Rotavirus, RV) is a double stranded RNA virus genome segment, to Reoviridae, rotavirus in the virus, caused by an important etiology and epidemiology of diarrhea in infants. The status of the mechanism of RV replication, especially on the regulation mechanism of autophagy after infecting the host cells and the virus The relationship between the replication capacity of in-depth and meticulous research. This research is not only to reveal the molecular mechanism of virus replication, screening has important significance to study the pathogenic mechanism and drug targets. In this study we adopted the method of small RNA deep sequencing, the wild strain ZTR68 isolated from endemic areas (G1P[8] (MA104) host cells infected monkey kidney cell line miRNAs) were differentially expressed after the discovery, the small molecules of RNA chr-1755 expression was significantly higher than the other, so that the molecule may play a major role in the replication of RV, named vsRNA1755., in order to confirm whether the molecular expression of universality in different virus and in the different cell lines were selected in 4 different type G (G2, G3, G4, G9) RV and two cells HT-29 (human colon cancer cell line), Caco-2 (human colorectal adenocarcinoma cell line) were By contrast, found that high vsRNA1755 expression. Then a series of regulatory mechanism of MA104 cells infected with G1 ZTR68 strain after done in-depth research. In early stage of viral replication found the relationship between the two positive ability and vsRNA1755 relationship. By comparing BLAST and NSP4 gene transfection in vitro analysis showed that the source of vsRNA1755 non structural protein in RV NSP4 (encoding enterotoxin and viral RNA replication related gene) gene, is composed of small molecular RNA NSP4 gene coil-coiled region encoding, participate in the biosynthesis of AGO1 by Dicer-1 and vsRNA1755. In order to find the downstream target genes, predicted by miRanda, revealing the insulin-like growth factor 1 receptor (Insulin-like Growth Factor 1 Receptor, IGF1R) is vsRNA1755 target. We used dual luciferase reporter system, after in vitro transfection of vsRNA1755 mimics (mimics) transfected cells to detect expression, certificate two There are targeted, vsRNA1755 can downregulate the expression of IGF1R. The target gene was obtained after our relationship, the biological functions of vsRNA1755 are discussed, considering that IGF1R is the starting element of PI3K/Akt pathway activation and autophagy pathway is closely related to our experimental research focusing on the mechanism of vsRNA1755 and autophagy detection reagent. Flow through the box (autophagy specific detection) detection and morphology, when transfected with autophagy marker protein LC3 re infection after RV, LC3-I converted to LC3-II, which indicated the phenomenon of autophagy specific RV infected cells. Co transfection of green fluorescent protein (EGFP) displays the results with LC3 tracer, and RV LC3 is Co located, to further verify the close relationship between RV infection and autophagy. The morphological observation of autophagosomes appeared 4 hours after infection, infection increased significantly, the latter form of the virus pool become visible Large number of virus particles increased. When the autophagy inhibitor 3-MA, decreased viral replication capacity, promote viral replication. Suggest that autophagy and autophagy regulation mechanism in vsRNA1755 experiment, we use the analogue of the molecule (mimics) transfected cells multiple protein autophagy regulation (ATG5, ATG12, P62). The expression was up-regulated the expression of autophagy related protein ATG5 and ATG12, down regulate the expression of autophagy substrates of P62. Western blot results showed that overexpression of vsRNA1755, autophagy specific substrates of P62 protein decreased obviously by adding PI3K/Akt inhibitor LY294002, detection of virus infection and copy number, results showed that the replication of the PI3K/Akt pathway on virus inhibition the results suggest that. QRT-PCR and Western blotting detection of vsRNA1755 and target gene IGF1R, RV expression in the early stage of infection can be reduced IGF1R, suggesting that vsRNA1755 inhibits PI3K/Akt/ through down-regulation of IGF1R The activation of mTOR signaling pathway, activation of autophagy, promote viral replication in vitro transfection. Mimics activation of this pathway results raised also confirms this possibility. In conclusion, RV infection of host cells by small molecule NSP4, RNA gene encoding RV 1755 (vsRNA1755), through inhibiting IGF1R expression induced by PI3K/Akt/mTOR signaling pathway inactivation of disinhibition on autophagy pathway, activation of autophagy, virus hijacks the autophagy pathway cells promoted their replication. The results of the study not only provides the main basis for revealing the mechanism of RV infection, as well as the interaction between virus and host cells and future targeted, provides a theoretical basis for meaningful the design of drug targets more accurately.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373

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