Biochemical and genetic evidence for complex formation between the influenza A virus NS1 protein and the interferon-induced PKR protein kinase
Biochemical and genetic evidence for complex formation between the influenza A virus NS1 protein and the interferon-induced PKR protein kinase
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DOI：
10.1089/jir.1998.18.757
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发表时间：
1998-09-01
影响因子：
2.3
通讯作者：
Katze, MG
中科院分区：
文献类型：
--
作者：
Tan, SL;Katze, MG
干扰素诱导的蛋白激酶（PKR）作为mRNA翻译起始的看门人发挥功能，因此是宿主IFN诱导的抗病毒防御系统的关键介质。许多病毒都有针对PKR的对策，有些病毒显然使用不止一种机制。流感病毒可以通过使用至少两种因子，细胞p58（IPK）蛋白和病毒NS1蛋白来抑制PKR活性。后者的确切作用方式尚未确定。在这里，使用共沉淀试验，我们发现，PKR可以形成一个复杂的从流感病毒感染的HeLa细胞制备的粗细胞提取物与NS1。利用酵母双杂交系统和体外结合实验验证了NS1与PKR的相互作用。缺失分析将NS1结合位点定位于PKR调控区N端98个残基。此外，NS1突变体，它缺乏PKR抑制活性，不结合PKR。最后，在PKR抑制中的功能作用的NS1的PKR活性的体内测定证实。这些结果支持NS1在病毒感染过程中PKR调节中的作用，该作用是通过两种蛋白质之间的复合物形成介导的。
The interferon (IFN)-induced protein kinase (PKR) functions as a gatekeeper of mRNA translation initiation and is, therefore, a key mediator of the host IFN-induced antiviral defense system. Many viruses have invested countermeasures against PKR, Some apparently use more than one mechanism. The influenza virus can repress PKR activity through the use of at least two factors, the cellular p58(IPK) protein and the viral NS1 protein. The exact mode of action of the latter has not been established. Here, using a coprecipitation assay, we found that PKR could form a complex with NS1 in crude cell extracts prepared from influenza virus-infected HeLa cells. The NS1-PKR interaction was verified by using the yeast two-hybrid system and an in vitro binding assay. Deletion analysis mapped the NS1 binding site to the N-terminal 98 residues of PKR regulatory region. Furthermore, an NS1 mutant, which lacks PKR inhibitory activity, did not bind PKR, Finally, the functional role of NS1 in PKR inhibition was substantiated using an in vivo assay for PKR activity. These results support the role of NS1 in PKR modulation during viral infection that is mediated through a complex formation between the two proteins.