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.

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

“…Notably, a lipid-binding pocket is also formed by the membrane-associated helices of the envelope glycoproteins E1 and E2 on the surface of mature alphaviruses, with a tryptophan (conserved among mosquito-borne alphaviruses) being involved in the stabilization of this site [45]. [11,12] in mature TBEV (A), Spondweni virus (B), dengue virus serotype 2 (C), and Japanese encephalitis virus (D). Color code: E protein domain I, red; E protein stem, green; transmembrane (TM) domains, gray.…”

“…Our findings are in agreement with the loss of infectivity of dengue virus serotype 2 particles with a W420A mutation (W421A in TBEV) [ 12 ], although detailed experiments for identifying the reasons of this defect were not performed with the dengue mutant. As revealed by cryo-electron microscopy (EM) structure analyses, CS connects two helices of the stem as a loop, both in mature and immature particles [ 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 44 ]. In mosquito-borne flaviviruses, W421 constitutes the last residue of H2, whereas in TBEV it is the first residue of the loop ( Figure 1 B and Figure 9 ).…”

“…The E glycoprotein is an elongated molecule consisting of three external domains (DI, DII, DIII), connected to a double membrane anchor by the so-called stem. 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 only amino acid in the CS that is conserved not only across tick-borne and mosquito-borne flaviviruses but also across those with unknown vectors (no-known vector flaviviruses [ 18 ]) is residue W421 (TBE virus, TBEV, numbering, Figure 1 B). Recent high-resolution structures of mature Zika, Spondweni and dengue viruses revealed pockets involving the stem of E that can accommodate lipids (pocket factors) [ 10 , 11 , 12 ]. These sites have been hypothesized as being crucial for stabilizing mature virus particles [ 10 , 11 , 12 ] and are located on either side of H1, with the CS being involved in the site that is closer to the viral membrane (site 1 as defined in reference [ 12 ], see also Figure 1 B).…”

“…Recent high-resolution structures of mature Zika, Spondweni and dengue viruses revealed pockets involving the stem of E that can accommodate lipids (pocket factors) [ 10 , 11 , 12 ]. These sites have been hypothesized as being crucial for stabilizing mature virus particles [ 10 , 11 , 12 ] and are located on either side of H1, with the CS being involved in the site that is closer to the viral membrane (site 1 as defined in reference [ 12 ], see also Figure 1 B). In our work, we addressed the role of W421 more specifically and investigated its influence on virus formation and/or entry.…”