Song Yuqing, Liu Xuechun, Che Yongjun, Wang Jinlong, Tian Zhancheng, Guan Guiquan, Roy Polly, Yin Hong, Du Junzheng
State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou, China.
J Virol. 2025 Aug 7:e0089625. doi: 10.1128/jvi.00896-25.
Bluetongue virus (BTV) is an arbovirus (genus, ; family, ) transmitted among ruminant hosts by biting midges (genus, ). . produce large numbers of virus-specific tubules during infection. Non-structural protein 1 (NS1) derived from BTV is a highly conserved tubule-forming protein that exists in infected cells as tubular and non-tubular forms. However, the lack of tagged viruses has limited dynamic tracking of NS1 tubule protein levels in living cells. Here, four recombinant viruses (BTV1-NS1-156TC, BTV1-NS1-156eGFP, BTV1-NS1-534eGFP, and BTV1-NS1-552eGFP) were successfully rescued using a BTV reverse genetics strategy by introduction of biarsenical-tetracysteine or enhanced green fluorescent protein (eGFP) tags to the BTV NS1 protein. Inserting these tags at these three sites resulted in different equilibrium states of NS1 between the tubular and non-tubular forms. NS1 was tracked in live cells to observe the dynamic movement and aggregation processes of NS1 tubules. The formation of NS1 tubules is a prerequisite for NS1 aggregation, and NS1 tubules are dependent on cellular microtubules to aggregate at the microtubule organizing center and encapsulate by vimentin to form aggresomes. This study confirms the possibility of controlling the conversion ratio between NS1 monomers and tubule forms, for the first time elucidates the movement and aggregation processes of NS1 tubules by live-cell imaging, and confirms that NS1 tubules form aggresomes.IMPORTANCEWhile extensive research has established BTV as a model system to study large non-enveloped viruses, critical gaps remain in our understanding of its biology. During infection, BTV NS1 assembles into abundant tubulars, a hallmark feature of the genus. In this study, fluorescently tagged BTV NS1 proteins were engineered, and four recombinant viruses were successfully rescued. Insertion of these tags altered the equilibrium between tubular and monomeric NS1 conformations, demonstrating the feasibility of modulating the transition ratio between structural states. Additionally, NS1 tubules depend on microtubule-mediated intracellular transport for aggregation and subsequent aggresome formation. These findings provide a basis for the application of NS1 in vaccine delivery, therapy, nanotechnology, and future explorations of BTV pathogenesis.
蓝舌病病毒（BTV）是一种虫媒病毒（属，；科，），通过叮咬蠓（属，）在反刍动物宿主之间传播。在感染期间会产生大量病毒特异性小管。源自BTV的非结构蛋白1（NS1）是一种高度保守的形成小管的蛋白，以管状和非管状形式存在于受感染细胞中。然而，缺乏标记病毒限制了对活细胞中NS1小管蛋白水平的动态追踪。在此，通过将双砷四硫氨酸或增强型绿色荧光蛋白（eGFP）标签引入BTV NS1蛋白，利用BTV反向遗传学策略成功拯救了四种重组病毒（BTV1-NS1-156TC、BTV1-NS1-156eGFP、BTV1-NS1-534eGFP和BTV1-NS1-552eGFP）。在这三个位点插入这些标签导致NS1在管状和非管状形式之间呈现不同的平衡状态。在活细胞中追踪NS1以观察NS1小管的动态运动和聚集过程。NS1小管的形成是NS1聚集的先决条件，并且NS1小管依赖于细胞微管在微管组织中心聚集并被波形蛋白包裹以形成聚集体。本研究证实了控制NS1单体与小管形式之间转化率的可能性，首次通过活细胞成像阐明了NS1小管的运动和聚集过程，并证实NS1小管形成聚集体。重要性虽然广泛的研究已将BTV确立为研究大型无包膜病毒的模型系统，但我们对其生物学的理解仍存在关键差距。在感染期间，BTV NS1组装成大量小管，这是该属的一个标志性特征。在本研究中，构建了荧光标记的BTV NS1蛋白，并成功拯救了四种重组病毒。这些标签的插入改变了管状和单体NS1构象之间的平衡，证明了调节结构状态之间转变比率的可行性。此外，NS1小管依赖于微管介导的细胞内运输进行聚集以及随后的聚集体形成。这些发现为NS1在疫苗递送、治疗、纳米技术以及未来对BTV发病机制的探索中的应用提供了基础。
