Flavivirus maturation leads to the formation of an occupied lipid pocket in the surface glycoproteins

Abstract

Flaviviruses such as Dengue (DENV) or Zika virus (ZIKV) assemble into an immature form within the endoplasmatic reticulum (ER), and are then processed by furin protease in the trans-Golgi. To better grasp maturation, we carry out cryo-EM reconstructions of immature Spondweni virus (SPOV), a human flavivirus of the same serogroup as ZIKV. By employing asymmetric localised reconstruction we push the resolution to 3.8 Å, enabling us to refine an atomic model which includes the crucial furin protease recognition site and a conserved Histidine pH-sensor. For direct comparison, we also solve structures of the mature forms of SPONV and DENV to 2.6 Å and 3.1 Å, respectively. We identify an ordered lipid that is present in only the mature forms of ZIKV, SPOV, and DENV and can bind as a consequence of rearranging amphipathic stem-helices of E during maturation. We propose a structural role for the pocket and suggest it stabilizes mature E.

Fig. 1. Cryo-EM structures of immature SPOV.

a Cryo-EM density map of immature SPOV (icosahedral symmetry applied). Bold numbers indicate threefold and fivefold vertices of the virion. The prM₃E₃ trimer of one asymmetric unit is colored (prM in blue, E in purple). Approximate dimensions of the virion are indicated by the accompanying scale bar. b Fourier shell correlation (FSC) plots of reconstructions using gold-standard refinement in RELION. Approximate map resolutions according to the 0.143 FSC cutoff are indicated for all reconstructions. Curves are shown for masked maps. c Cryo-EM density map of the prM₁E₁ complex at 3.8 Å resolution, obtained via localized reconstruction and focused refinement. The map served as the basis for building an initial atomic model. E is colored in purple, prM in blue. Locations of the furin protease recognition motif and fusion peptide (colored in red) are indicated. d Atomic model of the prM₃E₃ trimeric spike, refined against the 4.2 Å resolution map (orientation rotated by 90° relative to a). One copy of prM1E1 is shown in ribbon representation, with transmembrane (TM) helices indicated, and the two others as surfaces. The viral membrane is shown schematically. A cartoon depicting the organization of the trimer is shown for clarity. e Close-up of the area surrounding the furin recognition motif as indicated in c. E and prM residues involved in stabilizing the site are labeled. A schematic is included at the bottom of the panel showing the sequence after which furin cleaves pr and M.

Fig. 2. Anchoring of the prM linker to E.

a Zoomed out view of an E-prM complex. The boxed region indicates the location of the prM-H101 pocket. b Zoomed-in view of the boxed region indicated in a. A hydrophobic pocket on the surface of E is shown, into which prM-H101 is inserted. Involved E and prM residues are labeled. c Multiple sequence alignment (MSA) of flaviviral prM sequences. The positions of the conserved prM-H101 (SPOV numbering) are indicated, as well as the upstream furin site. SPONV Spondweni virus, ZIKV Zika virus, DENV Dengue virus, WNV West Nile virus, SLEV Saint Louis encephalitis virus, JEV Japanese encephalitis virus, YFV Yellow Fever virus, POWV Powassan virus, TBEV Tick-borne encephalitis virus.

Fig. 3. 2.6 Å resolution cryo-EM structure of mature SPOV.

a Cryo-EM density map of mature SPOV. Bold numbers indicate threefold and fivefold vertices of the virion. Each icosahedral asymmetric unit (ASU) contains three copies of E (colored brown, purple, and green for one ASU) and three copies of M, concealed below the E proteins. Approximate dimensions of the virion are indicated by the accompanying scale bar. b Top-view and side-view of an antiparallel dimer of E, which compose the surface of the mature virus. One copy is depicted in ribbon representation (purple), whereas for the other copy the cryo-EM map is shown (green). The M protein is indicated in the side-view, as well as a schematic of the viral membrane. A surface-exposed loop containing the glycan site is labeled. c Close-up view showing the density map of the glycan linked to N154 of E. There was clear density for the two first N-acetylglucosamines (GlcNac) and core fucosylation (Fuc). d Superposition of the pr-binding interface of E in mature and immature states. The comparison shows that a hairpin close to the pr-binding site of E shifts its position to better engage pr (immature hairpin in gray, mature in black). Residues on the hairpin involved in binding pr are shown as sticks and labeled.

Fig. 4. Formation of a lipid pocket by rearrangement of membrane-associated helices.

a Position and organization of membrane-associated helices in immature E. Two amphipathic helices lie flat on the membrane (colored green and red), whereas two transmembrane (TM) helices span it (colored yellow and orange). b Close-up view of the membrane-associated helices of immature E and mature E of SPOV. Amphipathic helices are numbered H1 and H2, TM helices TM1 and TM2. In mature E (on the right), the amphipathic helices have reorganized, opening up a pocket that is filled with density resembling a phosphatidylethanolamine (PE) lipid. The lipid is shown in stick representation. The density fit is shown separately in c for clarity. Residues in close vicinity are shown as sticks and labeled appropriately. c Lipid fitted into the cryo-EM map at the binding pocket. d MSA of E sequences located next to the lipid, highlighting the conservation of H447 and G451 (SPOV numbering). Virus abbreviations as in Fig. 2. e Density present in the pocket of mature ZIKV (accession code: EMDB-7543). f Density present in the pocket of mature DENV2 (this study). g Structure of Sindbis virus E1 and E2 (PDB-ID: 6IMM) illustrating the similarity of the position of a glycoprotein associated lipid (colored in red). h Virus recovery after Gibson assembly of mutant viruses. Wild-type and mutants of DENV2/16681 were constructed via Gibson assembly and propagated in C6/36 cells. Production of virus was assessed after given intervals by ELISA. Cells infected with virus stock served as positive control. The results of n = 2 biologically independent experiments are shown.