CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever

Abstract

Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional. The intracellular dimeric NS1 (iNS1) forms part of the viral replication complex. Previous studies suggest the extracellular secreted NS1 (sNS1), which is a major factor contributing to DHF, exists as hexamers. The structure of the iNS1 is well-characterised but not that of sNS1. Here we show by cryoEM that the recombinant sNS1 exists in multiple oligomeric states: the tetrameric (stable and loose conformation) and hexameric structures. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain – elongated β-sheet or β-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.

Structural insights into the distinct protective mechanisms of human antibodies targeting ZIKV NS1

“…In this study, we illustrated the epitopes of the five human anti-NS1 antibodies by solving the cryo-EM structures at atomic resolution. The purified sNS1 from S f 9 cells was found to mainly exist as a tetramer, consistent with the finding that the recombinant DENV2 sNS1 exists predominantly in the tetrameric form from 293F cell 28 . Our data provided detail on how five antibodies recognize the epitopes of multiple oligomeric sNS1 and the validation the function of antibodies.…”

Roles of NS1 Protein in Flavivirus Pathogenesis

“…Based on previous studies by Flamand et al, 1999, and Shu et al, 2022, the peak at around 300 kDa seen for WT NS1 is likely to correspond to the hexamer. Therefore, these observations imply that the I273 residue of NS1 is crucial for promoting higher-order oligomerization of the protein other than the dimers. , Tamura et al, 2022, further assessed the impact of the I273H mutation on the interaction of NS1 with membranes and observed that I273H did not alter the morphology of the liposomes, while the WT JEV NS1 was able to convert large liposomes into nanolipid structures, altering lipid membrane morphology, consistent with previous reports by Akey et al, 2014. , Therefore, it can be concluded that NS1 oligomerization into higher-order oligomers is critical for its secretion to cell culture supernatant and lipids’ membrane association . Another JEV NS1 mutant, F160D, which could not facilitate viral replication but retained the ability to secrete into the culture supernatant, demonstrated a similar capacity to modify lipid membranes as observed with the WT JEV NS1 .…”

“…As stated earlier, NS1 can exist in multiple oligomeric forms; it can exist as a membrane-associated dimer intracellularly (iNS1) and as a hexamer that gets secreted from the infected cells (sNS1) or remains associated with the cell surface via a GPI anchor …”

“…In a recent study, Shu et al, using cryo-EM, revealed that sNS1 in fact exists as a heterogeneous mixture of oligomers composed of tetramer (dimer of dimer) and hexamer (trimer of dimer) structures. The tetramers exist in two conformations: stable and loose.…”

“…Therefore, these observations imply that the I273 residue of NS1 is crucial for promoting higher-order oligomerization of the protein other than the dimers. 93,126 Tamura et al, 2022, further assessed the impact of the I273H mutation on the interaction of NS1 with membranes and observed that I273H did not alter the morphology of the liposomes, while the WT JEV NS1 was able to convert large liposomes into nanolipid structures, altering lipid membrane morphology, consistent with previous reports by Akey et al, 2014. 127,133 Therefore, it can be concluded that NS1 oligomerization into higher-order oligomers is critical for its secretion to cell culture supernatant and lipids' membrane association.…”