Stepwise assembly and mechanical stability of nsP3 Helical Scaffolds in Chikungunya Virus

Chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus that replicates its genome through a viral replication complex (RC) essential for successful infection. The RC consists of four non-structural proteins: nsP1, nsP2, nsP3, and nsP4. In this study, we focused on nsP3, which comprises three distinct domains: the alphavirus unique domain (AUD), the long and intrinsically disordered hypervariable domain (HVD), and the enzymatic macrodomain (MD). Notably, nsP3 assembles into helicoidal macrostructures, termed helical scaffolds (HSs), which are components of the CHIKV replication complex and are involved in the formation of macromolecular traps in the cytoplasm of the infected cell called Alphagranules (AGs). HSs form tubular helical scaffolds assembled through the AUD, with the MD and HVD flexibly deployed to the scaffold exterior. Despite their functional importance, the mechanisms governing HS formation remain poorly understood. Using High-Speed Atomic Force Microscopy (HS-AFM) and Electron Microscopy (EM) we achieved new findings on the structure, mechanics and assembly of these macrostructures. Our results show the nsP3 monomers follow intermediate steps before completing the assembly into full helical scaffolds, with each nsP3 domain contributing differently to this process. The macrodomain (MD) provides mechanical stability, yet it fractures under tens of piconewtons of force. This rigidity acts as a mechanical regulator that constrains the position of the HVD, which in turn mediates interactions with host factors and neighboring HSs. Through this interplay, nsP3 achieves both structural cohesion and functional adaptability, promoting the higher-order organization of HSs into honeycomb-like networks.