ABSTRACT

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.

摘要

由内在核定位信号（NLS）介导的胞质RNA病毒蛋白的核定位在成功的病毒复制中起重要作用。我们以前曾报道过，基质（M）蛋白中的NLS突变明显减弱了新城疫病毒（NDV）的复制和致病性，但减弱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，而且损害了病毒RNA的合成和转录。使用基于TMT的定量蛋白质组学分析感染了亲本NDV rSS1GFP和带有M / NLS突变的突变NDV rSS1GFP-M / NLSm感染的BSR-T7 / 5细胞，我们发现，与rSS1GFP-M / NLSm感染相比，rSS1GFP感染刺激了参与宿主细胞转录，核糖体结构，翻译后修饰和细胞内运输的差异表达蛋白的数量更大和表达变化更多。进一步的深入分析表明，M蛋白的主要核蓄积抑制了宿主细胞的转录，RNA的加工和修饰，蛋白合成，转录后的修饰和转运。当大多数M蛋白被限制在细胞核之外时，这种抑制作用可能会减弱。更重要的是，我们发现细胞质中M蛋白的功能以剂量依赖的方式抑制了TIFA表达，并通过下调TIFA / TRAF6 /NF-κB介导的细胞因子的产生促进了NDV复制。这是有关M蛋白参与NDV免疫逃逸的第一份报告。综上所述，我们的发现表明NDV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。