Structure and function of RNA viral interferon suppressor complexes

描述（由申请人提供）：该项目的长期目标是破译RNA病毒如何抑制宿主的先天免疫反应。短期目标是收集呼吸道合胞病毒（RSV）的两种独特的非结构（NS）蛋白如何抑制I型干扰素（IFN）的诱导和功能的生物化学和机制的理解，这使得病毒能够引起严重的下呼吸道感染和相关的死亡率。这些研究是基于我们最近的几个新发现：（i）RSV NS蛋白可以形成NS 1-NS 2异聚体。(ii)在它们之间，NS 1和NS 2降解IFN诱导和应答途径的多个成员，如RIG-I、IKK、IRF 3、IRF 7、TRAF 3和STAT 2。(iii)通过从重组表达NS 1或NS 2或两者的A549细胞中分离提取物，我们分离了以下主要成分的三种不同的复合物，这取决于表达哪种NS：（1）NS 1复合物或NS 1C：NS 1，Elongin BC，Cullin 2，Rbx 1;（2）NS 2复合物或NS 2C：NS 2，Elongin BC，Cullin 5，Rbx 2;（3）双重NS 1/NS 2复合物或NS 1/2C：NS 1、NS 2、Elongin BC、Cullin 2 & 5、Rbx 1 & 2、BAG 2、RAD 23 B。因此，NS 1/2C似乎是NS 1C和NS 2C的异源二聚体，但含有两个额外的宿主蛋白，BAG 2和RAD 23 B。(v)我们已经开发了所有三个NS复合物的体外测定，表明它们具有E3连接酶活性。(vi)这些IFN相关底物募集至NS复合物的特异性似乎由ISG 15缀合决定。(vii)我们意外地发现NS 1是一种磷蛋白，可能被共济失调毛细血管扩张突变（ATM）家族蛋白激酶磷酸化，这为NS 1激活开辟了一条新途径。在这个AREA（R15）项目中，我们将追求两个特定的目标，其中我们将：测试NS复合物中E3连接酶组分的作用;验证NS 1和NS 2的推定BC盒结构域;询问ISG化在NS E3连接酶底物特异性中的作用;探索辅助亚基的作用。（BAG 2，RAD 23 B）和NS 1和BAG 2的磷酸化;最后，通过构建和使用重组磷酸化NS 1突变体RSV在小鼠模型中验证NS 1的磷酸化在体内的作用。总之，我们的研究结果应该提供新的分子见解如何病毒劫持宿主细胞泛素-蛋白酶体降解系统的功能。它也可能为未来针对这些复合物的抗病毒方案提供框架。最后，它将为CSU的学生提供一个跳板，学习蛋白质-蛋白质相互作用，泛素化和蛋白质磷酸化的各个方面，特别是先天免疫的病毒抑制。

DESCRIPTION (provided by applicant): The long-term goal of this project is to decipher how RNA viruses suppress the host's innate immune response. The short-term goal is to glean a biochemical and mechanistic understanding how the two unique nonstructural (NS) proteins of respiratory syncytial virus (RSV) operate to suppress both the induction and function of type I interferon (IFN), which enables the virus to cause severe lower respiratory tract infection and associated mortality. The proposed studies are founded on several novel observations that we have made recently: (i) RSV NS proteins can form NS1-NS2 heteromer. (ii) Between them, NS1 and NS2 degrade multiple members of both IFN induction and response pathways, such as RIG-I, IKK¿, IRF3, IRF7, TRAF3 and STAT2. (iii) By fractionating extracts from A549 cells in which either NS1, or NS2, or both were expressed recombinantly, we isolated three distinct complexes of the following major constituents, depending on which NS was expressed: (1) NS1 Complex or NS1C: NS1, Elongin BC, Cullin 2, Rbx1; (2) NS2 Complex or NS2C: NS2, Elongin BC, Cullin 5, Rbx2; (3) the dual NS1/NS2 Complex or NS1/2C: NS1, NS2, Elongin BC, Cullin 2 & 5, Rbx1 & 2, BAG2, RAD23B. Thus, NS1/2C appears to be a heterodimer of NS1C and NS2C, but contains two additional host proteins, BAG2 and RAD23B. (v) We have developed in vitro assays for all three NS complexes, showing that they possess E3 ligase activity. (vi) The specificity of recruitment of these IFN-related substrates to the NS complexes seems to be determined by ISG15 conjugation. (vii) We made the unexpected discovery that NS1 is a phosphoprotein, likely phosphorylated by an Ataxia telangiectasia mutated (ATM) family protein kinase, which opens a novel avenue of NS1 activation. In this AREA (R15) project, we will pursue two Specific Aims, in which altogether we will: test the role of the E3 ligase components in the NS complexes; validate the putative BC box domain of NS1 and NS2; interrogate the role of ISGylation in NS E3 ligase substrate specificity; explore the role of accessory subunits (BAG2, RAD23B) and the phosphorylations of NS1 and BAG2; and lastly, validate the role of phosphorylation of NS1 in vivo by constructing and using recombinant phosphosite NS1 mutant RSV in the mouse model. Together, our results should provide new molecular insights into how a virally hijacked host cellular ubiquitin-proteasomal degradation system functions. It may also offer a framework for future antiviral regimen targeting these complexes. Finally, it would offer our students at CSU a springboard to learn various aspects of protein-protein interaction, ubiquitination and protein phosphorylation, with special reference to viral suppression of innate immunity.