Plum Island Animal Disease Center, Agricultural Research Service, USDA, Greenport, New York, USA
Plum Island Animal Disease Center, Agricultural Research Service, USDA, Greenport, New York, USA.
J Virol. 2019 Dec 12;94(1). doi: 10.1128/JVI.01642-19.
The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.
经典猪瘟病毒（CSFV）的 E2 蛋白是主要的病毒结构糖蛋白，是病毒粒子的重要组成部分。E2 已被证明参与了几种功能，包括病毒吸附、诱导保护性免疫和猪的毒力。我们之前使用酵母双杂交系统，鉴定了一种猪宿主蛋白 dynactin 亚基 6（DCTN6）（细胞 dynactin 复合物的组成部分）作为 E2 的特定结合伴侣。我们通过另外两种独立的方法，即共免疫沉淀和邻近连接分析，证实了 CSFV 感染的猪细胞中 DCTN6 和 E2 蛋白之间的相互作用。通过使用随机突变的 E2 文库的反向酵母双杂交方法，绘制了 E2 残基介导与 DCTN6 蛋白-蛋白相互作用的图谱。开发了携带这些关键残基特定取代的重组 CSFV 突变体 E2ΔDCTN6v，以评估 E2-DCTN6 蛋白-蛋白相互作用对病毒复制和猪病毒毒力的重要性。与亲本病毒相比，CSFV E2ΔDCTN6v 在建立的猪细胞系（SK6）和原代猪巨噬细胞培养物中的复制能力降低。值得注意的是，感染 CSFV E2ΔDCTN6v 的动物在 21 天的观察期内保持临床正常，这表明 CSFV E2 在感染过程中有效地与宿主 DCTN6 蛋白结合的能力可能在病毒毒力中起作用。结构糖蛋白 E2 是 CSFV 的重要组成部分，因为它参与了许多病毒活动，特别是病毒-宿主相互作用。在这里，我们介绍了病毒感染过程中 E2 与猪宿主蛋白 DCTN6 之间的蛋白-蛋白相互作用的描述和特征。还确定了介导与 DCTN6 相互作用的 E2 氨基酸残基。构建了携带突变破坏 E2-DCTN6 相互作用的重组 CSFV。使用反向遗传学研究了破坏 E2-DCTN6 蛋白-蛋白相互作用的影响。结果表明，破坏 E2-DCTN6 相互作用的相同氨基酸取代构成了天然宿主家猪中病毒毒力的关键因素。这突出了 E2-DCTN6 蛋白-蛋白相互作用在 CSFV 毒力中的潜在重要性，并为开发改进的 CSF 疫苗提供了可能的病毒减毒机制。
