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

## TL;DR

This study uses advanced imaging to reveal the 3D structure of virus protein filaments in live cells, showing they form a complex web rather than simple lines.

## Contribution

The use of genetic-code expansion and live-cell 3D-SIM to visualize RVFV NSs filaments in real time with superresolution.

## Key findings

- NSs filaments form a micron-scale polygon web with distinct domains in live-cell nuclei.
- Fixed-cell experiments confirmed the polygon web structure of NSs filaments.
- The polygon architecture supports structural plasticity and functional importance during infection.

## Abstract

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, J. Gen. Virol.
34, 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.