Live-cell 3D-SIM of Rift Valley fever virus NSs filaments reveals a polygon 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 [R. Swanepoel, N. K. Blackburn, <i>J. Gen. Virol.</i> <b>34</b>, 557-561 (1977).], as judged from fixed specimens. To gain insight into the 3D structure of NSs filaments within live-cell nuclei, we used genetic-code expansion (GCE) to incorporate trans-cyclooct-2-en-L-lysine into the protein. This enabled site-specific fluorescent labeling with tetrazine dyes for live-cell structured illumination microscopy (SIM). Our superresolved images revealed the complete native architecture of NSs filaments as a micron-scale polygon web of fibers with discrete domain characteristics, overturning previous assumptions of simple linear filaments. Parallel experiments on fixed RVFV-infected cells confirmed that native NSs filaments also display this morphology. Overall, our 3D-SIM analysis reveals distinct structural plasticity within NSs filaments, establishing a quantitative structure-function relationship that support the importance of polygon organization for NSs filament function during RVFV infection.