State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology, and the Research Center of Infection and Immunology, The University of Hong Kong, Hong Kong SAR, China.
J Virol. 2012 Dec;86(23):12695-707. doi: 10.1128/JVI.00647-12. Epub 2012 Sep 12.
The nonstructural protein (NS1) of influenza A virus performs multiple functions in the virus life cycle. Proteomic screening for cellular proteins which interact with NS1 identified the cellular protein RAP55, which is one of the components of cellular processing bodies (P-bodies) and stress granules. To verify whether NS1 interacts with cellular P-bodies, interactions between NS1, RAP55, and other P-body-associated proteins (Ago1, Ago2, and DCP1a) were confirmed using coimmunoprecipitation and cellular colocalization assays. Overexpression of RAP55 induced RAP55-associated stress granule formation and suppressed virus replication. Knockdown of RAP55 with small interfering RNA (siRNA) or expression of a dominant-negative mutant RAP55 protein with defective interaction with P-bodies blocked NS1 colocalization to P-bodies in cells. Expression of NS1 inhibited RAP55 expression and formation of RAP55-associated P-bodies/stress granules. The viral nucleoprotein (NP) was found to be targeted to stress granules in the absence of NS1 but localized to P-bodies when NS1 was coexpressed. Restriction of virus replication via P-bodies occurred in the early phases of infection, as the number of RAP55-associated P-bodies in cells diminished over the course of virus infection. NS1 interaction with RAP55-associated P-bodies/stress granules was associated with RNA binding and mediated via a protein kinase R (PKR)-interacting viral element. Mutations introduced into either RNA binding sites (R38 and K41) or PKR interaction sites (I123, M124, K126, and N127) caused NS1 proteins to lose the ability to interact with RAP55 and to inhibit stress granules. These results reveal an interplay between virus and host during virus replication in which NP is targeted to P-bodies/stress granules while NS1 counteracts this host restriction mechanism.
甲型流感病毒的非结构蛋白 (NS1) 在病毒生命周期中发挥多种功能。通过蛋白质组学筛选与 NS1 相互作用的细胞蛋白，鉴定出细胞蛋白 RAP55，它是细胞加工体 (P 体) 和应激颗粒的组成部分之一。为了验证 NS1 是否与细胞 P 体相互作用，使用免疫共沉淀和细胞共定位实验证实了 NS1、RAP55 和其他 P 体相关蛋白 (Ago1、Ago2 和 DCP1a) 之间的相互作用。RAP55 的过表达诱导 RAP55 相关应激颗粒形成并抑制病毒复制。用小干扰 RNA (siRNA) 敲低 RAP55 或表达与 P 体缺陷相互作用的显性负突变 RAP55 蛋白，阻止 NS1 与 P 体共定位。NS1 的表达抑制 RAP55 的表达和 RAP55 相关 P 体/应激颗粒的形成。在没有 NS1 的情况下，发现病毒核蛋白 (NP) 被靶向应激颗粒，但当 NS1 共表达时，NP 定位到 P 体。通过 P 体限制病毒复制发生在感染的早期阶段，因为随着病毒感染的进行，细胞中与 RAP55 相关的 P 体数量减少。NS1 与 RAP55 相关 P 体/应激颗粒的相互作用与 RNA 结合有关，并通过蛋白激酶 R (PKR) 相互作用的病毒元件介导。引入 RNA 结合位点 (R38 和 K41) 或 PKR 相互作用位点 (I123、M124、K126 和 N127) 的突变使 NS1 蛋白失去与 RAP55 相互作用的能力，并抑制应激颗粒。这些结果揭示了病毒复制过程中病毒与宿主之间的相互作用，在此过程中 NP 被靶向到 P 体/应激颗粒，而 NS1 则抵消了这种宿主限制机制。
