猪瘟病毒NS5A蛋白诱导细胞自噬的分子机理研究

Classical swine fever virus (CSFV) can induce cell autophagy and utilize autophagy to promote its proliferation. NS5A is an important multifunctional non-structural protein of CSFV. We confirmed for the first time that NS5A interacts with autophagy related proteins and can activate the autophagy marker LC3 in cells, suggesting that NS5A protein may play an important role in inducing autophagy. This project will establish porcine testicular cell lines that exogenously express NS5A protein, and systematically research the effect of NS5A protein on cell autophagy at the cellular and molecular level to clarify the changes in the expression of autophagy related molecules. To explore the functional regions and key amino acid sites for NS5A protein induced autophagy; CO-IP, GST-Pulldown and confocal laser scanning confocal microscope were used to study the protein interaction between NS5A and autophagy marker LC3. The role of LC3 protein played in NS5A-induced autophagy was studied by lentivirus-mediated gene knockout technique. This project will deeply reveal the role of NS5A protein in inducing autophagy and its molecular mechanism, providing a new scientific basis for elucidation of the mechanism of CSFV-induced autophagy.

猪瘟病毒（CSFV）能诱发细胞自噬，并利用自噬来促进其增殖。NS5A是CSFV的一个重要的多功能非结构蛋白。我们首次证实NS5A与自噬相关蛋白存在相互作用，并且能引起细胞中自噬标志物LC3的活化，提示NS5A蛋白可能在诱发细胞自噬方面发挥重要作用。本项目将建立在猪睾丸细胞中外源表达NS5A蛋白的细胞系，在细胞及分子水平系统研究NS5A蛋白对细胞自噬的影响，明确自噬相关分子表达变化情况；探究NS5A蛋白诱发细胞自噬过程中发挥作用的功能区及关键氨基酸位点；利用CO-IP、GST-Pulldown及激光共聚焦技术研究NS5A与自噬标志物LC3之间的蛋白相互作用；进一步通过慢病毒介导基因敲除技术研究LC3在NS5A诱发细胞自噬中所扮演的角色。本课题将深入揭示NS5A蛋白在诱发细胞自噬中的作用及其分子机制，为阐明CSFV诱发细胞自噬的机制提供新的科学依据。