# Zika NS2B Protein: In Vitro Formation of Large Multimeric Networks

## TL;DR

The Zika virus NS2B protein forms large multimers in both detergent and lipid environments, which may help in viral replication and membrane reshaping during infection.

## Contribution

This study reveals the complex oligomerization behavior of ZIKV NS2B, forming large multimers that were previously uncharacterized.

## Key findings

- ZIKV NS2B forms multimers ranging from dimers to over 10 subunits in detergent and lipid environments.
- AlphaFold predicted a monomeric model of NS2B but did not confidently predict oligomeric structures.
- NS2B multimerization may aid in membrane destabilization and reshaping during viral infection.

## Abstract

Flaviviruses are responsible for significant morbidity and mortality worldwide. Despite intensive research, the structure and oligomerization properties of non-structural (NS) proteins, like NS2 or NS4, are still uncertain because of their high hydrophobicity. Solution NMR has shown that NS2B protein has two hydrophobic domains, organized as two short α-helical hairpins that contribute to both viral RNA replication and particle formation. These are separated by a hydrophilic loop that is a cofactor of protease NS3. However, the oligomerization behavior of NS2B has not been explored in detail. Herein, we have expressed Zika virus NS2B protein (ZIKV NS2B) and characterized its oligomerization in both detergent and lipids using crosslinking in liposomes, and mass photometry and analytical ultracentrifugation in detergent. We show that, in contrast to the small oligomers proposed earlier, ZIKV NS2B protein has a very complex oligomerization behavior, forming from dimers to very large multimers (>10) in both detergent and lipids. Although AlphaFold (AF) provided a model for monomeric NS2B that is consistent with available experimental data, no oligomeric model was predicted with confidence. We suggest that the role of the two short α-helical hairpins in membrane destabilization and reshaping host ER during viral infection may be aided or triggered by multimerization. Finally, although our results report a high tendency of NS2B to oligomerize, in the context of the infected cell, a biologically relevant multimeric complex may necessitate other viral proteins like NS4A or NS4B and/or host proteins.