Structure and function of RNA viral interferon suppressor complexes

描述(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。短期目标是收集生化和机制了解呼吸道合胞病毒(RSV)的两种独特的非结构(NS)蛋白是如何工作的，以抑制I型干扰素(IFN)的诱导和功能，I型干扰素使病毒能够导致严重的下呼吸道感染和相关死亡。提出的研究基于我们最近的几个新的观察结果：(I)RSV NS蛋白可以形成NS1-NS2异构体。(Ii)在它们之间，NS1和NS2降解了干扰素诱导和反应通路的多个成员，如RIG-I、IKK、IRF3、IRF7、TRAF3和STAT2。(3)NS1/NS2复合体或NS1/2C复合体：NS1、NS2、Elongin BC、Cullin 2&5、Rbx2、RAD23B。因此，NS1/2C似乎是NS1C和NS2C的异源二聚体，但含有另外两个宿主蛋白bag2和RAD23B。(V)我们建立了三个NS复合体的体外检测方法，表明它们都具有E3连接酶活性。(Vi)这些干扰素相关底物向NS复合体募集的特异性似乎由ISG15结合决定。(Vii)我们有一个意想不到的发现，NS1是一种磷化蛋白，可能被毛细血管扩张共济失调突变(ATM)家族蛋白激酶磷酸化，这开启了NS1激活的新途径。在这一领域(R15)项目中，我们将追求两个特定的目标，其中我们总共将：测试E3连接酶组件在NS复合体中的作用；验证NS1和NS2假定的BC盒结构域；询问ISG化在NS E3连接酶底物专一性中的作用；探索辅助亚单位(bag2、RAD23B)和NS1和bag2的磷酸化的作用；最后，通过构建和使用重组亚磷酸盐NS1突变体RSV在小鼠模型中验证NS1的磷酸化作用。总之，我们的结果应该为病毒劫持的宿主细胞泛素-蛋白酶体降解系统如何发挥作用提供新的分子见解。这也可能为未来针对这些复合体的抗病毒方案提供一个框架。最后，它将为我们在CSU的学生提供一个跳板，学习蛋白质相互作用、泛素化和蛋白质磷酸化的各个方面，特别是关于病毒抑制先天免疫的问题。

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.