Extensive flavivirus E trimer breathing accompanies stem zippering of the post‐fusion hairpin

Abstract

Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod-like E subunits bend into "hairpins" to bring the fusion loops next to the C-terminal transmembrane (TM) anchors, with the TM-proximal "stem" element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E-protein trimers from the tick-borne encephalitis flavivirus with sequential stem truncations. Our thermostability and antibody-binding data suggest that the stem "zipper" ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C-terminally truncated upstream the CS, which, unlike fully stem-zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important "breathing" intermediates in the E-protein-driven membrane fusion process.

An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles

“…Likewise, the mutants were still capable of displaying those acidic-pH-triggered conformational changes that convert metastable E dimers into stable trimers and are necessary for membrane fusion during virus entry. These data are consistent with experiments that showed that the stem region is not involved in the stabilization of the E-trimer [13] and a study with a W421I SVP mutant of TBEV that was not only able to form stable post-fusion trimers, but also to fuse efficiently with liposomes [25].…”

“…This structural element consists of two or three mostly amphipathic helices (H1, H2, H3) [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ] and is located between the E-ectodomain and the viral membrane ( Figure 1 A). Two regions in E are highly conserved, the fusion loop (FL) at the tip of DII and the so-called conserved sequence in the stem (CS, amino acids 420–425 in TBEV, [ 13 ]) that forms a loop between H2 and H3 ( Figure 1 A). The FL is crucial for mediating fusion during virus entry after virus uptake by receptor-mediated endocytosis [ 14 ], but the role of CS is less clearly defined.…”

“…Upon virus entry into host cells via endocytosis, the low pH of the endosome triggers a massive structural reorganization of E that mediates membrane fusion and results in the conversion of E dimers into trimers [ 2 , 14 ]. The residues of the stem upstream of CS have been shown to stabilize the trimer during this process [ 13 ].…”

Implications of Dengue Virus Maturation on Vaccine Induced Humoral Immunity in Mice

“…The conserved linear fusion loop epitope (FLE) is readily exposed on the immature structure and is poorly presented on the mature virion [ 18 ]. The mature virion undergoes dynamic E dimer ‘breathing’, a process in which the tip of EDII hinges at the EDI–EDII interface at biological temperatures, causing increased fusion loop exposure [ 19 , 20 , 21 ]. Antibodies to the fusion loop epitope are often weakly cross-neutralizing and are broadly cross-reactive [ 22 , 23 ].…”

Impact of structural dynamics on biological functions of flaviviruses

“…Only one of the three stem-anchor regions in the trimer is shown in panels (C) and (D).Colour code E as in Figs.1 and 2. Molecular structures shown in panel A were generated with PyMol (https://pymol.org/2) using coordinates of TBEV (PDBs: 1URZ, 6S8C[27,31]).…”