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, Jiaxiu South Road, Huaxi District, Guiyang, 550025, Guizhou, China.
Vet Res. 2020 Sep 22;51(1):120. doi: 10.1186/s13567-020-00846-1.
Bromodomain-containing protein 2 (BRD2) is a nucleus-localized serine-threonine kinase that plays pivotal roles in the transcriptional control of diverse genes. In our previous study, the chicken BRD2 (chBRD2) protein was found to interact with the Newcastle disease virus (NDV) matrix (M) protein using a yeast two-hybrid screening system, but the role of the chBRD2 protein in the replication of NDV remains unclear. In this study, we first confirmed the interaction between the M protein and chBRD2 protein using fluorescence co-localization, co-immunoprecipitation and pull-down assays. Intracellular binding studies indicated that the C-terminus (aa 264-313) of the M protein and the extra-terminal (ET) domain (aa 619-683) of the chBRD2 protein were responsible for interactions with each other. Interestingly, although two amino acids (T621 and S649) found in the chBRD2/ET domain were different from those in the human BRD2/ET domain and in that of other mammals, they did not disrupt the BRD2-M interaction or the chBRD2-M interaction. In addition, we found that the transcription of the chBRD2 gene was obviously decreased in both NDV-infected cells and pEGFP-M-transfected cells in a dose-dependent manner. Moreover, small interfering RNA-mediated knockdown of chBRD2 or overexpression of chBRD2 remarkably enhanced or reduced NDV replication by upregulating or downregulating viral RNA synthesis and transcription, respectively. Overall, we demonstrate for the first time that the interaction of the M protein with the chBRD2 protein in the nucleus promotes NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. These results will provide further insight into the biological functions of the M protein in the replication of NDV.
溴结构域蛋白 2（BRD2）是一种定位于细胞核的丝氨酸/苏氨酸激酶，在多种基因的转录调控中发挥关键作用。在我们之前的研究中，利用酵母双杂交筛选系统发现鸡 BRD2（chBRD2）蛋白与新城疫病毒（NDV）基质（M）蛋白相互作用，但 chBRD2 蛋白在 NDV 复制中的作用尚不清楚。在本研究中，我们首先通过荧光共定位、共免疫沉淀和下拉实验证实了 M 蛋白与 chBRD2 蛋白之间的相互作用。细胞内结合研究表明，M 蛋白的 C 端（aa264-313）和 chBRD2 蛋白的额外末端（ET）结构域（aa619-683）负责相互作用。有趣的是，尽管 chBRD2/ET 结构域中发现的两个氨基酸（T621 和 S649）与人类 BRD2/ET 结构域以及其他哺乳动物的 BRD2/ET 结构域不同，但它们并没有破坏 BRD2-M 相互作用或 chBRD2-M 相互作用。此外，我们发现 chBRD2 基因的转录在 NDV 感染细胞和 pEGFP-M 转染细胞中均明显降低，且呈剂量依赖性。此外，通过 chBRD2 小干扰 RNA 介导的敲低或 chBRD2 的过表达，分别上调或下调病毒 RNA 合成和转录，显著增强或降低了 NDV 的复制。总之，我们首次证明，M 蛋白与核内 chBRD2 蛋白的相互作用通过下调 chBRD2 表达并促进病毒 RNA 合成和转录来促进 NDV 复制。这些结果将进一步深入了解 M 蛋白在 NDV 复制中的生物学功能。
