PDF(Pubmed)
Abstract：
The interaction between viral proteins and host proteins plays a crucial role in the process of virus infecting cells. Tags such as HA, His, and Flag do not interfere with the function of fusion proteins and are commonly used to study protein-protein interactions. Adding these tags to viral proteins will address the challenge of the lack of antibodies for screening host proteins that interact with viral proteins during infection. Obtaining viruses with tagged fusion proteins is crucial. This study established a new reverse genetic system with T7 promoter and three plasmids, which efficiently rescued Newcastle disease virus (NDV) regardless of its ability to replicate in cells. Subsequently, using this system, NDV containing a HA-tagged structural protein and NDV carrying a unique tag on each structural protein were successfully rescued. These tagged viruses replicated normally and exhibited genetic stability. Based on tag antibodies, every NDV structural protein was readily detected and showed correct subcellular localization in infected cells. After infecting cells with NDV carrying HA-tagged M protein, several proteins interacting with the M protein during the infection process were screened using HA tag antibodies. The establishment of this system laid the foundation for comprehensive exploration of the interaction between NDV proteins and host proteins.
摘要：
病毒蛋白与宿主蛋白之间的相互作用在病毒感染细胞的过程中起着至关重要的作用。标签如HA,他的,和Flag不干扰融合蛋白的功能，通常用于研究蛋白质-蛋白质相互作用。将这些标签添加到病毒蛋白将解决缺乏用于筛选在感染期间与病毒蛋白相互作用的宿主蛋白的抗体的挑战。获得带有标记的融合蛋白的病毒至关重要。本研究建立了一个新的具有T7启动子和三个质粒的反向遗传系统，无论其在细胞中复制的能力如何，都能有效地拯救新城疫病毒（NDV）。随后，使用这个系统，成功挽救了含有HA标记的结构蛋白的NDV和在每个结构蛋白上携带独特标签的NDV。这些标记的病毒正常复制并表现出遗传稳定性。基于标签抗体，每个NDV结构蛋白都很容易检测到，并在感染细胞中显示正确的亚细胞定位。用携带HA标记的M蛋白的NDV感染细胞后，使用HA标签抗体筛选了在感染过程中与M蛋白相互作用的几种蛋白。该系统的建立为全面探索NDV蛋白与宿主蛋白的相互作用奠定了基础。
