Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education, Guizhou University , Guiyang, China.
College of Animal Science, Guizhou University , Guiyang, China.
Virulence. 2020 Dec;11(1):607-635. doi: 10.1080/21505594.2020.1770482.
Nuclear localization of cytoplasmic RNA virus proteins mediated by intrinsic nuclear localization signal (NLS) plays essential roles in successful virus replication. We previously reported that NLS mutation in the matrix (M) protein obviously attenuates the replication and pathogenicity of Newcastle disease virus (NDV), but the attenuated replication mechanism remains unclear. In this study, we showed that M/NLS mutation not only disrupted M's nucleocytoplasmic trafficking characteristic but also impaired viral RNA synthesis and transcription. Using TMT-based quantitative proteomics analysis of BSR-T7/5 cells infected with the parental NDV rSS1GFP and the mutant NDV rSS1GFP-M/NLSm harboring M/NLS mutation, we found that rSS1GFP infection stimulated much greater quantities and more expression changes of differentially expressed proteins involved in host cell transcription, ribosomal structure, posttranslational modification, and intracellular trafficking than rSS1GFP-M/NLSm infection. Further in-depth analysis revealed that the dominant nuclear accumulation of M protein inhibited host cell transcription, RNA processing and modification, protein synthesis, posttranscriptional modification and transport; and this kind of inhibition could be weakened when most of M protein was confined outside the nucleus. More importantly, we found that the function of M protein in the cytoplasm effected the inhibition of TIFA expression in a dose-dependent manner, and promoted NDV replication by down-regulating TIFA/TRAF6/NF-κB-mediated production of cytokines. It was the first report about the involvement of M protein in NDV immune evasion. Taken together, our findings demonstrate that NDV replication is closely related to the nucleocytoplasmic trafficking of M protein, which accelerates our understanding of the molecular functions of NDV M protein.
细胞质 RNA 病毒蛋白通过固有核定位信号 (NLS) 的核定位在病毒成功复制中起着至关重要的作用。我们之前报道过，基质 (M) 蛋白中的 NLS 突变明显削弱了新城疫病毒 (NDV) 的复制和致病性，但衰减的复制机制仍不清楚。在这项研究中，我们表明 M/NLS 突变不仅破坏了 M 的核质运输特性，还损害了病毒 RNA 合成和转录。使用 TMT 基于定量蛋白质组学分析感染亲本 NDV rSS1GFP 和携带 M/NLS 突变的突变 NDV rSS1GFP-M/NLSm 的 BSR-T7/5 细胞，我们发现 rSS1GFP 感染刺激了更多数量和更多表达变化的与宿主细胞转录、核糖体结构、翻译后修饰和细胞内运输相关的差异表达蛋白，而 rSS1GFP-M/NLSm 感染则不然。进一步深入分析表明，M 蛋白的主要核积累抑制了宿主细胞转录、RNA 加工和修饰、蛋白质合成、转录后修饰和运输；并且当大部分 M 蛋白被限制在核外时，这种抑制可以被削弱。更重要的是，我们发现 M 蛋白在细胞质中的功能以剂量依赖的方式影响 TIFA 表达的抑制，并通过下调 TIFA/TRAF6/NF-κB 介导的细胞因子产生来促进 NDV 复制。这是关于 M 蛋白参与 NDV 免疫逃避的首次报道。总之，我们的研究结果表明，NDV 的复制与 M 蛋白的核质运输密切相关，这加速了我们对 NDV M 蛋白分子功能的理解。
