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。

The interaction between the host cell and the virus is important to reveal the mechanism of proliferation of the virus. In the process of viral infection, the virus can encode the expression of the microRNA molecule to regulate its genes and host genes, promoting the replication of the virus and avoiding the host's immune surveillance. Research shows that in the process of viral infection, the virus can hijack the autophagy membrane system of the host cell to replicate the virus itself. There are several regulatory processes and factors involved in the process, but the exact mechanism of the hijacking is not yet clear. Rotavirus (Rotavirus, RV) is a genetic component segments of double-stranded RNA virus, is called enterovirus, Rotavirus, in infants and young children diarrhea caused by has important status in etiology and epidemiology. Papers around the RV replication mechanism, especially to infected host cells after the regulation mechanism of autophagy and the relationship of viral replication ability carried on the thorough careful research. This study is of great significance not only to reveal the molecular mechanism of virus replication, but also to investigate the mechanism of pathogenesis and drug target screening.

In this study we adopt the method of the depth of the small RNA sequencing, the wild strains isolated from endemic areas ZTR68 (G1P [8]) infected host cells MA104 batches (monkey kidney cell line) after the screening of differentially expressed miRNAs, found the small RNA molecules chr-1755 expression is significantly higher than the other, thus speculated that the molecule may play a major role in the RV replication, named it the vsRNA1755. To confirm the molecule in different type of virus and whether exist in different cells expressing universal, experiment to choose the other four different types G (G2, and G3, G4, G9) of the RV and two types of cells HT-29 optimization colon cancer cell line (people), Caco-2 (cancer of colorectal adenomas) were compared, found vsRNA1755 all have high expression. A series of regulatory mechanisms after the G1 type ZTR68 strain infected MA104 cells were studied. The correlation was found in the virus's early replication ability and the vsRNA1755 relationship. Through Blsat comparison and in vitro transfection NSP4 gene analysis shows that vsRNA1755 comes from the RV nonstructural protein NSP4 enterotoxin (encoding genes associated with viral RNA replication) genes, are encoded by NSP4 gene coil- coiled area small RNA molecules, biosynthesis and AGO1 participation by Dicer -1.

To find the downstream target gene of vsRNA1755, a miRanda software forecast reveals that the insulin-like Growth Factor 1 Receptor, IGF1R, is a potential target for vsRNA1755. We use dual luciferase report system, by in vitro transfection vsRNA1755 and using vsRNA1755 simulation objects (mimics) expression in transfection cell detection, prove the existence of the relationship between target and cut IGF1R vsRNA1755 can express.

After get the relationship between the target genes, we on vsRNA1755 biological function are discussed in this paper, considering the IGF1R is the starting component of PI3K/Akt pathway and closely related to the activation of autophagy pathway, we experiment on vsRNA1755 and the mechanism of autophagy research. By autophagy flow detection kit (specificity) and electron microscopy morphology inspection, found that when the transfection autophagy protein markers LC3 again after the RV infection, LC3 - I converted to LC3 - II, that have taken place in the RV infection of cells after clear autophagy. The tracer results of green fluorescent protein (EGFP) and LC3 showed that RV and LC3 had a co-located relationship, which further validates that RV infection is closely related to autophagy. Morphological observations showed that after infection, the infection occurred in small bodies, with a significant increase in the number of infections between 4 and 6 hours, and the subsequent increase in the number of virus particles. When the virus replicates and the virus replicates, the virus replication is prompted by the use of autophagy 3 - MA. Autophagy regulatory mechanism in research vsRNA1755 experiment, we use the molecular simulation objects (mimics) transfection cells tested multiple autophagy regulatory proteins (ATG5, ATG12, P62) expression, found that autophagy related proteins ATG5 and ATG12 expression, autophagy substrate P62 expression. The protein-printing experiment showed that after expressing vsRNA1755, the autophagy specific substrate P62 protein was significantly reduced.

By adding the PI3K/Akt pathway inhibitor, LY294002, to detect the infection and copy number of the virus, the results show that the PI3K/Akt pathway inhibits the replication of the virus. To vsRNA1755 with target gene IGF1R qRT- PCR and protein imprinting testing, the results suggest that the expression of the RV early in infection can cut IGF1R, speculation vsRNA1755 cut IGF1R through inhibition of PI3K/Akt/mTOR signaling pathway activation, trigger, activate autophagy, to achieve viral replication. The results of the pathway were also confirmed by the increase in external transfection mimics.

To sum up, the RV infection after the host cell, by the RV NSP4 gene encoding of small RNA molecules (vsRNA1755)1755, caused by inhibiting IGF1R express PI3K/Akt/mTOR signaling pathways downstream of the deactivation, lift its inhibition of autophagy pathways, activate cell autophagy, viruses hijack a cell autophagy pathway to promote its own replication. Research results not only provides the main basis for revealing the RV infection mechanism, as well as interaction between virus and host cells and future targeted drug targets, more accurate design provides the theory basis of meaningful.