MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Department of Infectious Diseases and Public Health, City University of Hong Kong, Hong Kong SAR, China.
J Virol. 2021 Feb 24;95(6). doi: 10.1128/JVI.01928-20.
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine with high morbidity and mortality that negatively affects the pig industry worldwide, in particular in China. Soon after the endocytosis of CSFV, the virus makes full use of the components of host cells to complete its life cycle. The endocytosis sorting complex required for transport (ESCRT) system is a central molecular machine for membrane protein sorting and scission in eukaryotic cells that plays an essential role in many physiological metabolic processes, including invasion and egress of envelope viruses. However, the molecular mechanism that ESCRT uses to regulate the replication of CSFV is unknown. In this study, we demonstrated that the ESCRT-I complex Tsg101 protein participates in clathrin-mediated endocytosis of CSFV and is also involved in CSFV trafficking. Tsg101 assists the virus in entering the host cell through the late endosome (Rab7 and Rab9) and finally reaching the lysosome (Lamp-1). Interestingly, Tsg101 is also involved in the viral replication process by interacting with nonstructural proteins 4B and 5B of CSFV. Finally, confocal microscopy showed that the replication complex of Tsg101 and double-stranded RNA (dsRNA) or NS4B and NS5B protein was close to the endoplasmic reticulum (ER), not the Golgi, in the cytoplasm. Collectively, our finding highlights that Tsg101 regulates the process of CSFV entry and replication, indicating that the ESCRT plays an important role in the life cycle of CSFV. Thus, ESCRT molecules could serve as therapeutic targets to combat CSFV infection. CSF, caused by CSFV, is a World Organization for Animal Health (OIE) notifiable disease and causes significant financial losses to the pig industry globally. The ESCRT machinery plays an important regulatory role in several members of the genera and within the family , such as hepatitis C virus, Japanese encephalitis virus, and dengue virus. Previous reports have shown that assembling and budding of these viruses require ESCRT. However, the role of ESCRT in infection remains to be elucidated. We determined the molecular mechanisms of the regulation of CSFV infection by the major subunit Tsg101 of ESCRT-I. Interestingly, Tsg101 plays an essential regulatory role in both clathrin-mediated endocytosis and genome replication of CSFV. Overall, the results of this study provide further insights into the molecular function of ESCRT-I complex protein Tsg101 during CSFV infection, which may serve as a molecular target for pestivirus inhibitors.
经典猪瘟（CSF）是由经典猪瘟病毒（CSFV）引起的一种高传染性猪病，发病率和死亡率高，对全球养猪业造成负面影响，特别是在中国。CSFV 进入细胞后，会充分利用宿主细胞的成分完成其生命周期。内吞体分选复合物运输所需（ESCRT）系统是真核细胞中膜蛋白分选和分裂的核心分子机器，在包括包膜病毒入侵和出芽在内的许多生理代谢过程中发挥着重要作用。然而，ESCRT 调节 CSFV 复制的分子机制尚不清楚。在这项研究中，我们证明 ESCRT-I 复合物 Tsg101 蛋白参与 CSFV 的网格蛋白介导的内吞作用，并参与 CSFV 的运输。Tsg101 通过晚期内体（Rab7 和 Rab9）协助病毒进入宿主细胞，最终到达溶酶体（Lamp-1）。有趣的是，Tsg101 还通过与 CSFV 的非结构蛋白 4B 和 5B 相互作用参与病毒复制过程。最后，共聚焦显微镜显示，Tsg101 和双链 RNA（dsRNA）或 NS4B 和 NS5B 蛋白的复制复合物在细胞质中靠近内质网（ER），而不是高尔基体。总之，我们的发现强调了 Tsg101 调节 CSFV 进入和复制的过程，表明 ESCRT 在 CSFV 生命周期中发挥重要作用。因此，ESCRT 分子可以作为对抗 CSFV 感染的治疗靶点。CSF 是由 CSFV 引起的，是世界动物卫生组织（OIE）报告的疾病，给全球养猪业造成了重大经济损失。ESCRT 机械在家族中的 和 属的几个成员中发挥重要的调节作用，如丙型肝炎病毒、日本脑炎病毒和登革热病毒。以前的报告表明，这些病毒的组装和出芽需要 ESCRT。然而，ESCRT 在 感染中的作用仍有待阐明。我们确定了 ESCRT-I 主要亚基 Tsg101 调节 CSFV 感染的分子机制。有趣的是，Tsg101 在 CSFV 的网格蛋白介导的内吞作用和基因组复制中都发挥着重要的调节作用。总的来说，这项研究的结果进一步深入了解了 ESCRT-I 复合蛋白 Tsg101 在 CSFV 感染过程中的分子功能，这可能成为抗 Pestivirus 抑制剂的分子靶点。
