Live-cell 3D-SIM of Rift Valley fever virus NSs filaments reveals a polygonal web architecture

A defining feature of Rift Valley fever virus (RVFV) is the incorporation of the NSs protein into large filamentous assemblies inside infected nuclei 1, as judged from fixed specimens. To gain insight into the three-dimensional (3D) structure of NSs filaments within nuclei of live-cells we used genetic-code expansion (GCE) to introduce an unnatural amino acid (uAA) fluorophore into the protein, and coupled this with live-cell structured illumination microscopy (SIM). Our super-resolved images revealed a micron-scale polygonal web of NSs fibres with discrete domain characteristics. Parallel experiments on fixed RVFV-infected cells confirmed that virally-encoded native NSs filaments also display this morphology. Overall, our 3D-SIM analysis sheds new light on the complex large-scale architecture of NSs filaments and provides context for programmable filamentous E3 ligases that promote virus replication. SignificanceThis work demonstrates how advanced labeling and imaging techniques can reveal unexpected organizational principles in viral protein assemblies. We bio-orthogonally labeled the Rift Valley fever virus NSs protein with bright tetrazine dyes using genetic-code expansion (GCE), that enabled imaging of NSs filaments in live-cells using 3D-SIM. GCE offers key advantages for studying small, genetically fragile viral proteins like NSs. It eliminates the need for bulky fluorescent proteins that can disrupt protein-protein interfaces critical for complex assembly and avoids fixation artifacts associated with traditional immunofluorescence microscopy. This integrative GCE and 3D-SIM approach revealed the complete native architecture of NSs filaments in 3D, dramatically enhancing our ability to visualize intricate polygonal web structures beyond simple linear filaments. The discrete structural domains identified here raise intriguing questions about potential mechanisms for spatially controlling E3 ligase activity in a programmable manner.