TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein

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.

胞质RNA病毒蛋白通过固有核定位信号（nuclear localization signal，NLS）介导的核定位过程，对病毒的成功复制至关重要。本团队此前研究发现，基质蛋白（matrix protein，M）上的NLS突变可显著减弱新城疫病毒（Newcastle disease virus，NDV）的复制能力与致病性，但其复制减弱的具体分子机制仍不明确。本研究证实，M蛋白/NLS突变不仅破坏了M蛋白的核质穿梭特性，还损害了病毒RNA的合成与转录过程。采用基于串联质量标签（Tandem Mass Tag，TMT）的定量蛋白质组学分析，对感染亲本NDV rSS1GFP以及携带M/NLS突变的突变株NDV rSS1GFP-M/NLSm的BSR-T7/5细胞进行检测后发现，相较于rSS1GFP-M/NLSm感染组，rSS1GFP感染组可诱导更多数量的差异表达蛋白，且这些参与宿主细胞转录、核糖体结构、翻译后修饰以及胞内运输的差异蛋白的表达变化幅度更为显著。进一步的深度分析表明，M蛋白的主导性核积累会抑制宿主细胞的转录、RNA加工与修饰、蛋白质合成、转录后修饰以及运输过程；而当大部分M蛋白被限制于细胞核外时，这种抑制作用会被削弱。更为关键的是，本研究发现细胞质中的M蛋白可通过剂量依赖的方式抑制TIFA的表达，并通过下调TIFA/TRAF6/NF-κB介导的细胞因子产生来促进NDV的复制。本研究首次报道了M蛋白参与NDV免疫逃逸的现象。综上，本研究结果证实NDV的复制与M蛋白的核质穿梭特性密切相关，这一发现加深了我们对NDV M蛋白分子功能的理解。