PI3K/AKT在蝙蝠源呼肠孤病毒复制中的作用研究

Bat as reservoir hosts for a variety of zoonotic disease virus, plays an important role in the spread of infectious diseases. Although most people infected with the reovirus presented with asymptomatic or mild symptoms pass, but recent foreign reports indicate that bat-origin reovirus can spread from person to person and cause serious respiratory diseases. The bat-origin reovirus in this study is closed with serum type Ⅰstrains from phylogenetic analysis, but the mice suffered from severe disease with 100% mortality in the virus infection. In order to understand the mechanism of the PI3K/AKT signaling pathway regulating viral replication, A549 cells were infected as a model to evaluate the PI3K/AKT activation time; secondly explore PI3K/AKT activation pathway (depending on the recognition of viruses and cells, endocytosis, virus protein and/or viral replication) as well as the impact on viral RNA replication and protein translation. Viral infection-induced apoptosis at early is not benefit to the proliferation of the virus. There is no related research that viral replication capacity was enhanced through the activation of the PI3K/AKT which is to regulate or inhibit apoptosis-related genes in host cells by the synthesis or phosphorylation of proteins. This study aims to assure the role of PI3K/AKT signaling pathway in the infection and replication of bat-origin reovirus across the host, which can provide scientific support for the prevention of newly emerging infectious diseases.

蝙蝠作为多种人畜共患病病毒的储存宿主，在传染病的流行中扮演了重要的角色。近些年国外研究报道显示蝙蝠源呼肠孤病毒可以在人与人之间传播，并导致严重的呼吸道疾病。本研究以蝙蝠源呼肠孤病毒为研究对象，序列分析显示与血清Ⅰ型毒株较近，病毒感染小鼠后可以导致100%死亡。为了解PI3K/AKT信号通路调节该病毒复制的机制，本研究主要以感染A549细胞为模型，评价PI3K/AKT激活时间；其次探索PI3K/AKT激活途径（是否依赖于病毒与细胞受体的相互识别、细胞内吞、病毒蛋白成分及病毒复制）以及该通路对病毒RNA复制和蛋白翻译的影响。病毒感染早期诱导细胞凋亡不利于病毒的增殖，对于该病毒通过激活PI3K/AKT来调节抑制凋亡相关基因的表达以对抗宿主细胞凋亡来增强病毒的复制能力尚无相关研究。本研究旨在解析PI3K/AKT信号通路在蝙蝠源呼肠孤病毒跨宿主感染和复制中的作用，为新发传染病的防治提供科学支撑。

本研究以两株不同血清型野生动物源MRV，3型毒株MPC/04和1型MRV毒株B/03感染A549细胞为模型。 结果表明MPC/04和B/03在感染A549细胞时，均能够激活宿主细胞的ISRE元件，促进IFITM1、ISG15和Viperin等ISGs的表达。此外，MPC/04和B/03感染不表达IFN-β基因的Vero细胞系同样能够激活其ISRE启动子。封闭A549细胞I型IFN及II型IFN受体，不影响MPC/04和B/03激活ISRE途径的能力。说明MRV感染激活ISRE启动子元件不依赖于经典的IFN/JAK/STAT1途径。为进一步探索MRV活化ISGs表达是否依赖于PI3K/Akt信号通路的分子机制，通过Western blot和RNA干扰等方法，分析MRV感染过程对PI3K/Akt通路的激活情况。MPC/04和B/03在感染早期能够激活A549、HEK 293T和Vero细胞内Akt 的磷酸化。PI3K特异性抑制剂LY294002和Wortmannin处理，及下调p85α亚基蛋白表达均能够抑制MPC/04诱导Akt的磷酸化。此外，Chlorpromazine抑制Clathrin内吞途径显著降低MPC/04和B/03诱导的Akt磷酸化程度，而Genistein抑制Caveolin内吞途径不影响MPC/04和B/03诱导的Akt磷酸化程度。siRNA 干扰A549细胞Clathrin表达显著影响MRV诱导的Akt磷酸化程度。MPC/04感染早期同样能够诱导FAK激酶的磷酸化，不能够引起Gab1的磷酸化。FAK激酶特异性抑制剂PP2处理能够阻断MRV感染诱导的FAK和Akt的活化，干扰FAK蛋白的表达及过表达FAK显性负性突变体均能够明显抑制MRV激活PI3K/Akt信号通路。表明MRV感染早期通过病毒Clathrin介导的内吞途径及FAK磷酸化，激活宿主PI3K/Akt信号通路。MPC/04和B/03感染早期能够诱导Akt依赖的EMSY磷酸化，过表达EMSY能够促进MPC/04和B/03的复制。PI3K抑制剂LY294002处理、干扰Akt表达及上调EMSY的表达均能够抑制MPC/04和B/03感染激活ISRE启动子元件，及上调IFITM1，Viperin和ISG15的表达。表明MRV通过PI3K/Akt信号通路磷酸化EMSY促进ISGs的表达。