Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus.
Functional characterization and proteomic analysis of the nucleocapsid protein of porcine deltacoronavirus.
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DOI：
10.1016/j.virusres.2015.06.013
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发表时间：
2015-10-02
期刊：
影响因子：
5
通讯作者：
Lee C
中科院分区：
文献类型：
--
作者：
Lee S;Lee C
PDCoV N蛋白作为非共价连接的寡聚体存在。PDCoV N主要分布在核仁中。N过表达对细胞生长无影响。PDCoV N显著改变了两种细胞伴侣蛋白GRP78和HSC 70。猪三角洲冠状病毒（PDCoV）是一种新发现的嗜肠型猪冠状病毒，可引起仔猪肠炎和腹泻。与其他冠状病毒一样，PDCoV通常含有4种主要结构蛋白：刺突蛋白（S）、包膜蛋白（E）、膜蛋白（M）和核衣壳蛋白（N）。其中，已知N蛋白是最丰富和多功能的病毒组分。因此，作为了解PDCoV生物学的第一步，本研究调查了组成型表达PDCoV N蛋白的稳定猪细胞系中宿主蛋白的功能特征和表达动力学。与其他冠状病毒的N蛋白类似，发现PDCoV N蛋白与自身相互作用形成非共价连接的寡聚体，并且主要定位于核仁。然后，我们通过蛋白质组学分析评估了在不同时间点表达N的PK（PK-PDCoV-N）细胞中蛋白质产生水平的改变。根据高分辨率双向凝胶电泳结果，初步发现PK-PDCoV-N细胞与对照PK细胞相比，共有43个蛋白质点差异表达。在这些斑点中，10个蛋白斑点显示出统计学显著的改变，包括8个上调和2个下调的蛋白斑点，并被挑选用于随后的蛋白质鉴定，通过基质辅助激光解吸/电离飞行时间质谱法后的肽质量指纹图谱。我们在这项研究中鉴定的受影响的细胞蛋白质被分为参与各种细胞过程的功能组，如细胞分裂，代谢，应激反应，蛋白质合成和运输，细胞骨架网络和细胞通讯。值得注意的是，发现热休克蛋白70家族的两个成员在PK-PDCoV-N细胞中上调。这些蛋白质组学数据将为PDCoV感染期间对N蛋白的特异性细胞反应提供见解。
The PDCoV N protein exists as non-covalently linked oligomers. PDCoV N is predominantly distributed in the nucleolus. N overexpression had no effect on cell growth. PDCoV N significantly altered two cellular chaperone proteins, GRP78 and HSC70. Porcine deltacoronavirus (PDCoV) is a newly discovered enterotropic swine coronavirus that causes enteritis and diarrhea in piglets. Like other coronaviruses, PDCoV commonly contains 4 major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. Among these, the N protein is known to be the most abundant and multifunctional viral component. Therefore, as the first step toward understanding the biology of PDCoV, the present study investigated functional characteristics and expression dynamics of host proteins in a stable porcine cell line constitutively expressing the PDCoV N protein. Similar to N proteins of other coronaviruses, the PDCoV N protein was found to interact with itself to form non-covalently linked oligomers and was mainly localized to the nucleolus. We then assessed alterations in production levels of proteins in the N-expressing PK (PK-PDCoV-N) cells at different time points by means of proteomic analysis. According to the results of high-resolution two-dimensional gel electrophoresis, a total of 43 protein spots were initially found to be differentially expressed in PK-PDCoV-N cells in comparison with control PK cells. Of these spots, 10 protein spots showed a statistically significant alteration, including 8 up-regulated and 2 down-regulated protein spots and were picked for subsequent protein identification by peptide mass fingerprinting following matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The affected cellular proteins that we identified in this study were classified into the functional groups involved in various cellular processes such as cell division, metabolism, the stress response, protein biosynthesis and transport, cytoskeleton networks and cell communication. Notably, two members of the heat shock protein 70 family were found to be up-regulated in PK-PDCoV-N cells. These proteomic data will provide insights into the specific cellular response to the N protein during PDCoV infection.
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