痘苗病毒宿主范围基因K1L和C7L拮抗干扰素的功能研究

Type I Interferons (IFNs) are cytokines that play a critical role in immune defense against infection and tumor formation. The cellular factors that mediate this defense are the products of interferon-stimulated genes (ISGs). Among more than 300 identified ISGs, only a few have been characterized with respect to antiviral activity. We recently discovered that vaccinia virus (VACV) K1 and C7 proteins inhibit type I IFNs-induced antiviral activities by antagonizing effectors induced by IFN. We further showed that none of the well-characterized IFN effectors is the target of K1and C7. Therefore, further studying the mechanism by which K1 and C7 antagonize IFN offers a unique opportunity for uncovering novel ISG that maybe critical for innate immune defense against many viruses. VACV is unique among viruses used as a good study model in that it encodes a large number of proteins for evading host immune responses. Host-range genes mainly include K1L and C7L in VACV are one of the most important strategies that the virus used to against the host anti-viral response. Deletion of K1L and C7L from VACV results in abortive replication of the virus in many cells and severe attenuation in mice, and this is the reason why NYVAC, a vaccine vector that is currently tested in AIDS vaccine development. However, the molecular mechanism by which K1 and C7 regulate poxvirus host tropism remains unsolved because of lacking a good cell based model and structure supports. One innovation is we have set up a viral model (vK1L-C7L-) and IFN-inducible Huh7 cells model. Another innovation of our study is we have the crystal structure of K1 and C7. Based on this unique model we performed a large-scale ISG screening using a lentiviral ISG expression library. We have screened 350 ISGs and identified IRF1 as an ISG that specifically inhibits the replication of vK1L-C7L- but not WT VACV. Although IRF1 turns out to be another inducer of the antiviral factors our studies have greatly reduced the number of potential candidates to 65 and provided IRF1 as a positive control in our search. We hypothesize that K1 and C7 antagonize novel IFN-inducible antiviral factors that are among the 65 ISGs. We will screen the remaining 65 ISGs for specific inhibitors of vK1L-C7L-. In addition, we will take a complementary approach of siRNA knocked. The structures of K1 and C7 show they both resemble structural scaffolds used for ligand binding. Therefore, our additional approach will be to identify the binding partners of K1 and C7 by using reversible in-cell crosslinking, affinity purification and quantitative mass spectrometry analysis. Identification of the host targets of K1 and C7 will reveal novel ISGs that maybe critical for innate immune defense against many viruses. Our studies will provide important clues to the controlling mechanism for poxvirus host tropism and fundamental knowledge for further improving VACV-based vaccine vector for infectious diseases and cancer.

发现新的抗病毒干扰素刺激因子--ISG为抗病毒治疗提供新方法，是当今世界病毒学研究的热点。我们发现痘苗病毒宿主范围蛋白K1和C7对I型干扰素引起的抗病毒效应有拮抗作用，而K1和C7的靶点是未知的ISG。本项目拟从已筛出的65个候选ISG中找出K1和C7拮抗的ISG。①使用重组病毒vK1L-C7L-和Huh7细胞模型，建立针对候选基因的cDNA表达文库和siRNA，过量表达和沉默目的基因，发现抑制病毒增殖的ISG。②使用Tet-on系统建立可调控表达目的蛋白的Huh7细胞系，定量分析目的基因的抑制作用。③晶体结构提示K1和C7具有与配体结合的特殊构象，拟采用多种亲和纯化，如TAP、SILAC、细胞内蛋白交联结合新的质谱分析技术，获得与之作用的蛋白，揭示宿主范围蛋白拮抗干扰素作用的机制。本研究将丰富病毒拮抗宿主反应的理论，为痘苗病毒载体的研发提供理论指导，为抗病毒治疗提供新方法。

本研究旨在发现新的痘病毒的抗病毒因子或抑制因子。已经发现痘病毒（Vaccina virus （VACV））进化产生的宿主范围蛋白K1和C7可以拮抗宿主蛋白SAMD9的抗病毒功能。另外，一些哺乳动物组织中还表达一种SAMD9蛋白的同源蛋白，即SAMD9L。在有些哺乳动物组织中只保留了这两种蛋白中的一个。本研究发现SAMD9L与SAMD9功能相似，是一个新的痘病毒抑制因子，SAMD9/SAMD9L这两个细胞抑制因子共同构成了抑制痘病毒感染的宿主屏障。由于小鼠天然缺失SAMD9, 而只保留了SAMD9L, 所以成为研究SAMD9L功能的最佳动物模型。虽然同为痘病毒抑制因子，SAMD9表现为组成性活性，而SAMD9L却表现为干扰素诱导性活性。利用Crispr-Cas9技术敲除人源组织细胞中的SAMD9，vK1L-C7L-便可以复制。如果要实现vK1L-C7L-在干扰素处理过的人源细胞中复制，则需要同时敲除SAMD9和SAMD9L。虽然在人源组织中SAMD9和SAMD9L可以被痘病毒编码的K1、 C7及其同源蛋白所拮抗，但是在一些以反刍动物为主要宿主且具有较窄宿主范围的痘病毒所编码的病毒蛋白C7（例如sheep poxvirus）则不能拮抗小鼠SAMD9L的抑制作用。我们的实验结果发现：（1）小鼠细胞中SAMD9L是vK1L-C7L-病毒的抑制因子。（2）小鼠动物模型中鼠类的SAMD9L抑制 vK1L-C7L- 复制和致病。（3）在小鼠模型中羊痘病毒的C7同源蛋白在拮抗小鼠SAMD9L抑制作用上存在缺陷。 （4）大多数痘病毒的C7同源蛋白均可以结合mSAMD9L，而羊痘病毒的功能缺陷是由于两个氨基酸的突变所引起的。（5）在人源组织中，SAMD9 和 SAMD9L分别是作为构成性和IFN诱导性vK1L-C7L-病毒抑制因子而存在的。(6) 不同痘病毒的C7同源蛋白可以拮抗hSAMD9L的抑制功能，但是羊痘病毒由于两个氨基酸的变异，只具有较弱拮抗功能。基于上述研究结果，我们发现SAMD9L与SAMD9是功能相似的宿主抑制因子，痘病毒只有拮抗了SAMD9/L的功能才能在宿主内复制与致病。我们同时也发现SAMD9/L存在宿主种属特异性，并且痘病毒在拮抗SAMD9/L的功能上也存在种属特异性，这一结果显示SAMD9/L构成了阻止痘病毒跨种属传播的种间屏障。该课题的深入研究有望使我们对痘