Liji Xie, Zhixun Xie^*,  Sheng Wang,  Xianwen Deng, Zhiqin Xie

Department of Biotechnology, Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, 51 Youai North Road, Nanning 530001, China

Abstract     The present study was conducted to determine whether avian reovirus (ARV) activates the phosphatidylinositol 3-kinase-dependent Akt (PI3K/Akt) pathway according to the PXXP or YXXXM motifs of σA and σNS proteins. Gene splicing by overlap extension PCR was used to change the PXXP or YXXXM motifs of the σA and σNS genes. Plasmid constructs that contain mutant σA and σNS genes were generated and transfected into Vero cells, and the expression levels of the corresponding genes were quantified according to immunofluorescence and Western blot analyses. The Akt phosphorylation (P-Akt) profile of the transfected Vero cells was examined by flow cytometry and Western blot. The results showed that the σA and σNS genes were expressed in the Vero cells, and P-Akt expression in the σA mutant groups (amino acids 110–114 and 114–117) was markedly decreased. The results indicated that the σA protein of ARV activates the PI3K/Akt pathway via the PXXP motif. The results of this study reveal the mechanisms by which ARV manipulates the cellular signal transduction pathways, which may provide new ideas for novel drug targets.

Keywords  Avian reovirus,  PI3K/Akt pathway, σA protein, σNS protein,  PXXP motif

禽呼肠孤病毒σA和σNS蛋白激活PI3K/Akt信号通路的研究

谢丽基，谢芝勋*，王盛，邓显文，谢志勤

（广西壮族自治区兽医研究所 广西兽医生物技术重点实验室， 南宁 530001）

摘要： 旨在探讨禽呼肠孤病毒（ARV）的σA和σNS蛋白，是否是通过PXXP或YXXXM基序来激活PI3K/Akt信号通路。本研究采用重叠延伸PCR的方法，将σA和σNS基因的PXXP或YXXXM基序进行突变后构建了重组质粒，并在Vero细胞中进行了表达。通过流式细胞术和Western-blot，检测转染后Vero细胞磷酸化Akt（P-Akt）的表达量，并与野生型σA和σNS蛋白激活的P-Akt表达量进行比较。结果显示，σA和σNS基因均得到了表达，σA基因的110-114和114-117位的PXXP基序突变后， Vero细胞P-Akt的表达量明显下降（图1）。上述研究结果表明，ARV的σA蛋白是通过PXXP基序来激活PI3K/Akt信号通路的。本研究从细胞信号转导角度揭示了ARV与宿主相互作用的机制，也为寻找抗ARV药物作用靶标提供了新的思路。

关键词：禽呼肠孤病毒；PI3K/Akt信号通路；σA蛋白；PXXP基序

图1 流式细胞术检测P-Akt的表达. (A): Negative control, (B): pcAGEN, (C): σA-pcAGEN, (D): σA 55-58位PXXP突变, (E): σA 65-69位YXXXM突变, (F): σA 110-114位PXXP突变, (G): σA 114-117位PXXP突变, (H): σA 200-203位PXXP突变, I: σA 207-211位YXXXM突变

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