猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

In order to explore the role and specific molecular mechanism of the host protein Beclin1 in the process of the non-structural protein NS5A of classical swine fever virus (CSFV) activating the autophagy response of cells, in this study, in ST cells infected with CSFV and expressing the NS5A protein, the qRT - PCR method was used to detect the changes in the expression of Beclin1 and factors related to the PI3K/Akt pathway. The interaction relationship between Beclin1 and NS5A was studied by using methods such as laser confocal microscopy, Co - IP and GST - pulldown. The effect of overexpressing or knocking down Beclin1 in ST cells on the replication of CSFV was studied. The results showed that when ST cells were infected with CSFV or exogenously expressed the NS5A protein, the transcriptional and protein expression levels of Beclin1 were significantly increased, and the expression levels of factors related to the PI3K/Akt pathway were positively correlated with it. In addition, the CSFV NS5A protein and the Beclin1 protein had co - localization and interaction in the cells. Finally, we found that overexpressing Beclin1 in the cells significantly promoted the replication of CSFV; conversely, after knocking down Beclin1 with siRNA, the activation of the PI3K/Akt pathway was inhibited, and the proliferation of CSFV showed a significant inhibitory effect. The above results indicate that the Beclin1 protein has a promoting effect on the replication of CSFV, and its mechanism is achieved by regulating the PI3K/Akt pathway through the interaction with NS5A.

为探究宿主蛋白Beclin1在猪瘟病毒（classical swine fever virus，CSFV）非结构蛋白NS5A激活细胞自噬反应过程中的作用及具体分子机制，本研究在感染CSFV及表达NS5A蛋白的ST细胞中，利用qRT-PCR方法检测Beclin1、PI3K/Akt通路相关因子表达变化情况。利用激光共聚焦、Co-IP及GST-pulldown等方法研究Beclin1与NS5A相互作用关系。通过在ST细胞中过表达或敲低Beclin1，研究其对CSFV复制的影响。结果表明，ST细胞感染猪瘟病毒或外源表达NS5A蛋白，Beclin1转录和蛋白表达水平均显著升高，且PI3K/Akt通路相关因子表达水平与之呈正相关。此外，CSFV NS5A蛋白与Beclin1蛋白在细胞中存在共定位且具有相互作用。最后，我们发现在细胞中过表达Beclin1，对CSFV复制起到明显促进作用；反之，利用siRNA敲低Beclin1后，抑制PI3K/Akt通路活化，CSFV增殖表现出明显抑制效应。以上结果表明，Beclin1蛋白对CSFV复制具有促进作用，其机制是通过与NS5A的相互作用调控PI3K/Akt通路来实现。