Flavivirus NS4B proteins do not form homodimers: discrepancies with an AlphaFold-based oligomeric model

In flaviviruses such as Dengue, Zika or West Nile virus (DENV, ZIKV or WNV), non-structural protein 4B (NS4B) participates in membrane remodeling during infection and is critical for virulence and host immunity. Despite the important roles and confirmed drug target status for NS4B, no detailed structure is available. Flavivirus NS4B proteins have five hydrophobic domains and have been proposed to form homodimers. Herein, we have used AlphaFold to explore its multimeric organization in a diverse set of five sequences of flaviviruses. AlphaFold correctly predicts the α-helical segments identified by solution NMR, but with a fold that differs from current models involving regular transmembrane helices. Oligomers of increasing size were evaluated using a combined AlphaFold score, with optimal value centered around decamers (n = 10). We tested this model experimentally using ZIKV NS4B protein. Biochemical and biophysical data produced an oligomer larger than the current dimeric model, at least pentameric, and possibly hexameric. Overall, our results suggest a 'regular' NS4B oligomer in ZIKV that is at least pentameric with five regular transmembrane helices. Although no evidence was found for the 'irregular' Alphafold-predicted model, it is intriguing that it is common to all sequences tested. Interference with the formation of an NS4B oligomer may be a general novel approach to treat flavivirus infection.