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。

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.