Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic β-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.

Yellow fever virus (YFV) infections can cause severe diseases in humans, resulting in mass casualties in Africa and the Americas each year. Secretory NS1 (sNS1) is thought to be used as a diagnostic marker of flavivirus infections, playing an essential role in the flavivirus life cycle, but little is known about the composition and structure of YFV sNS1. Here, we present that the recombinant YFV sNS1 exists in a heterogeneous mixture of oligomerizations, predominantly in the tetrameric form. The cryoEM structures show that the YFV tetramer of sNS1 is stacked by the hydrophobic interaction between β-roll domains and greasy fingers. According to the 3D variability analysis, the tetramer is in a semi-stable state that may contain multiple conformations with dynamic changes. We believe that our study provides critical insights into the oligomerization of NS1 and will aid the development of NS1-based diagnoses and therapies.

Zika virus (ZIKV) Non-structural protein 1 (NS1) plays an essential role in viral replication and immune evasion. Our understanding of the differential protective mechanism of NS1-targeting antibodies is limited. Here, we determined the cryoEM structures of ZIKV NS1 in complex with two group antibodies at 2.6-2.9Å. Group I antibodies (3G2 and 4B8) potently recognize cell surface form of NS1 and multiple oligomeric forms of NS1 by occupy the epitopes on outer surface of dimeric NS1. IgG and Fab from group I antibodies completely abrogate sNS1-mediated endothelial dysfunction in vitro. Group II antibodies (4F10, 2E11, and 14G5) recognize the previously reported epitopes in distal end of the β -ladder domain of monomeric NS1, and their blockade efficiency depends on the affinity with NS1 protein. These findings elucidate the correlation between the epitope recognition and the protective efficacy of anti-NS1 antibodies and highlight the distinct mechanisms of therapeutic potential of 3G2 and 4B8.