Yang Yanling, Xue Jia, Teng Qingyuan, Li Xiao, Bu Yawen, Zhang Guozhong
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
J Virol. 2021 Mar 10;95(7). doi: 10.1128/JVI.02087-20. Epub 2021 Jan 13.
We previously demonstrated that W proteins from different Newcastle disease virus (NDV) strains localize in either the cytoplasm (e.g., NDV strain SG10) or the nucleus (e.g., NDV strain La Sota). To clarify the mechanism behind these cell localization differences, we overexpressed W protein derived from four different NDV strains or W protein associated with different cellular regions in Vero cells. This revealed that the key region for determining W protein localization is 180-227aa. Further experiments found that there is a nuclear export signal (NES) motif in W protein 211-224aa. W protein could be transported into the nucleus via interaction with KPNA1, KPNA2, and KPNA6 in a nuclear localization signal-dependent manner, and W protein containing an NES was transported back to the cytoplasm in a CRM1-independent manner. Interestingly, we observed that the cytoplasm-localized W protein colocalizes with mitochondria. We rescued the NES-deletion W protein NDV strain rSG10-ΔW/W using an NDV reverse genetics system and found that the replication ability, virulence, and pathogenicity of an NDV strain were all higher when the W protein cellular localization was in the nucleus rather than the mitochondria. Further experiments revealed that W protein nuclear localization reduced the expression of IFN-β otherwise stimulated by NDV. Our research reveals the mechanism by which NDV W protein becomes localized to different parts of the cell and demonstrates the outcomes of nuclear or cytoplasmic localization both and , laying a foundation for subsequent functional studies of the W protein in NDV and other paramyxoviruses. In Newcastle disease virus (NDV), the W protein, like the V protein, is a nonstructural protein encoded by the P gene via RNA editing. Compared with V protein, W protein has a common N-terminal domain but a unique C-terminal domain. V protein is known as a key virulence factor and an important interferon antagonist across the family In contrast, very little is known about the function of NDV W protein, and this limited information is based on studies of the Nipah virus W protein. Here, we investigated the localization mechanism of NDV W protein and its subcellular distribution in mitochondria. We found that W protein localization differences impact IFN-β production, consequently affecting NDV virulence, replication, and pathogenicity. This work provides new insights on the differential localization mechanism of NDV W proteins, along with fundamental knowledge for understanding the functions of W proteins in NDV and other paramyxoviruses.
我们之前证明，来自不同新城疫病毒（NDV）毒株的W蛋白定位于细胞质（如NDV毒株SG10）或细胞核（如NDV毒株La Sota）。为阐明这些细胞定位差异背后的机制，我们在Vero细胞中过表达了源自四种不同NDV毒株的W蛋白或与不同细胞区域相关的W蛋白。这表明决定W蛋白定位的关键区域是180 - 227aa。进一步实验发现，W蛋白的211 - 224aa存在一个核输出信号（NES）基序。W蛋白可通过与KPNA1、KPNA2和KPNA6相互作用，以依赖核定位信号的方式转运至细胞核，而含有NES的W蛋白则以不依赖CRM1的方式转运回细胞质。有趣的是，我们观察到定位于细胞质的W蛋白与线粒体共定位。我们使用NDV反向遗传系统拯救了缺失NES的W蛋白NDV毒株rSG10 - ΔW/W，发现当W蛋白的细胞定位在细胞核而非线粒体时，NDV毒株的复制能力、毒力和致病性均更高。进一步实验表明，W蛋白的核定位降低了原本由NDV刺激产生的IFN - β的表达。我们的研究揭示了NDV的W蛋白定位于细胞不同部位的机制，并展示了核定位或细胞质定位的结果，为后续对NDV及其他副粘病毒中W蛋白的功能研究奠定了基础。在新城疫病毒（NDV）中，W蛋白与V蛋白一样，是P基因通过RNA编辑编码的非结构蛋白。与V蛋白相比，W蛋白有一个共同的N端结构域，但有一个独特的C端结构域。V蛋白是整个病毒家族中已知的关键毒力因子和重要的干扰素拮抗剂。相比之下，关于NDV的W蛋白的功能知之甚少，且这些有限的信息是基于对尼帕病毒W蛋白的研究。在这里，我们研究了NDV的W蛋白的定位机制及其在线粒体中的亚细胞分布。我们发现W蛋白的定位差异会影响IFN - β的产生，进而影响NDV的毒力、复制和致病性。这项工作为NDV的W蛋白的差异定位机制提供了新的见解，同时也为理解NDV及其他副粘病毒中W蛋白的功能提供了基础知识。
