Visualization and tracking of tubule-derived, fluorescent-labeled NS1 as a marker of bluetongue virus in living cells

Bluetongue virus (BTV) is an arbovirus (genus, Orbivirus; family, Sedoreoviridae) transmitted among ruminant hosts by Culicoides biting midges (genus, Culicoides). Orbivirus sp. 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 Orbivirus 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.