VISA介导的细胞抗病毒信号转导的深层研究

We have previously identified a mitochondrial antiviral signaling protein termed VISA (also known as MAVS ,IPS-1, or CARDIF) that plays a pivotal role in RIG-I mediated antiviral signaling. Previously published data by our group and others show that VISA contains an N-terminal CARD domain for interacting with RIG-I and RIG-I induced antiviral signaling cascades; a proline-rich domain, and a C-terminal transmembrane domain that inserts VISA into the outer mitochondrial membrane (OMM). VISA also contains one TRAF2 and two TRAF6 interacting motifs necessary for VISA-induced NF-κB activation. A few of other proteins, like TRAF3,TRAF5,FADD, TRADD, RIP1, and proteins residing on OMM, including MITA, WDR5, TOMO70, gC1qR, NLRX1 and MFN2 were also depicted to interact with VISA, positively or negatively regulating VISA mediated antiviral signaling. However, the exact spatio-temporal events surrounding VISA protein following virus infection are still under intense investigation. To better understand the mechanism of the VISA mediated antiviral signaling, more VISA associated partners need to be identified. We had attempted to identify VISA-interacting proteins by using full-length VISA as bait in yeast two-hybrid (Y2H) screening to explore more VISA's partners. However, only TRAF2, TRAF6 were identified due to the false negative. Compared with eukaryotic cells, the expressed VISA protein in a yeast cell has different characteristics in function via structure alteration or lack of post-translational modifications, resulting in the false negative results. To circumvent these limitations, truncated VISA protein that only contain the CARD domain, proline-rich region, transmembrane domain will be used for the Y2H. Four genes; DAAM1, APRT, TMBIM4,BAT3 were identified as VISA's new partners and negative regulators from a preliminary screening by using VISA(360-540) as a bait while another candidate, C1IN, was identified as a positive regulator by using CARD domain as a bait. We concluded that using a truncated VISA containing different domains instead of the full length VISA as bait in Y2H will lead to a strengthened application for identifying VISA's new partners. Eventually, more VISA interacting proteins will be identified. VISA protein mediated antiviral events following virus infection is a dynamic process. The components of VISA related signalosome may change spatio-temporally. Based on this, we will make different 293 cell lines in which full length and/or truncated VISAs will be stabily expressed. The cell lines will be infected with Sendai virus, collected at different time course, and then tandem affinity purification and protein mass spectrometry will be used to identify VISA's interacting proteins. By using these two different approaches, more VISA interacting proteins will eventually be identified. Therefore, the mechanisms of VISA related events following virus infection will be manifested more clearly.

我们报道了一个定位在线粒体上的蛋白VISA(MAVs),在RIG-I的抗病毒信号中起重要作用。为深入研究VISA的作用机理，有必要鉴定更多的VISA作用蛋白。用VISA的全长作诱饵酵母双杂交筛选时，得不到好的筛选结果。我们改用含VISA不同功能域的短截体作诱饵并在筛选预实验中获得成功。用VISA(360-540)和VISA(1-110)为诱饵的筛选中，我们得到了DAAM1等4个与VISA相关并在其通路中起负调节作用的基因及一个能与VISA的CARD域作用并在抗病毒通路中起正调节作用的C1IN。这说明用VISA短截体作诱饵筛选是成功的。病毒感染细胞后，VISA介导的抗病毒反应是一个动态的，VISA的作用蛋白在这个过程中不断变化。我们将在病毒感染细胞后的不同时间点收集细胞，用串联亲和纯化方法纯化与VISA作用蛋白。对更多VISA作用蛋白的发现和功能研究，将进一步阐明VISA的作用机理。

VISA（Virus-Induced Signaling Adapter）是细胞内RIG-I样受体抗病毒信号通路重要的接头蛋白。上游RIG-I样受体在病毒入侵时被激活，VISA通过其CARD结构域结合活化的RIG-I样受体并发生寡聚化，活化的VISA招募E3泛素连接酶等相互作用蛋白激活下游信号通路，诱导I型干扰素表达。VISA除定位于线粒体外，还与过氧化物酶体、内质网及自噬体都有关联，在抗病毒信号通路与炎症反应途径中都重要作用，并参与细胞凋亡和代谢功能。由于其在细胞生命活动的重要作用，但仍然有许多的机制不太完善。为进一步完善VISA的功能机理，需要发现更多的相互作用蛋白提供理论基础。因此采用分子克隆的方法构建诱饵质粒pGBT9-VISA(1-540)，利用酵母双杂交技术从cDNA细胞文库中筛选VISA的相互作用蛋白，并利用免疫共沉淀、双荧光素酶报告系统等技术研究其相关功能。结果发现，成功筛选得到的HAUS8、ZWINT、GNB2L1等10个基因表达的蛋白与VISA具有相互作用，其中HAUS8和ZWINT可以增强由SeV介导的IRF3磷酸化，诱导IFN-β表达，参与激活VISA的抗病毒信号通路；而GNB2L1负调控VISA介导的抗病毒信号通路。