A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses

Abstract

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, we present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. We use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 Å, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. We anticipate the chimeric platform to be widely applicable for investigating flavivirus biology.

Morphological Diversity and Dynamics of Dengue Virus Affecting Antigenicity

“…Most of their periphery residues are involved in making contact with adjacent E proteins either to form a dimer (intradimer), interdimer or inter-raft interactions, leaving no gaps on the virus surface. Due to this compactness, mature virus structures have been solved to a higher resolution compared to the immature virus structures—DENV1 (4.5 Å, 4CCT) [ 14 ], DENV2 (3.1 Å, 6ZQU), DENV3 (6 Å, 3J6S) [ 53 ], DENV4 (4.1 Å, 4CBF) [ 51 ], ZIKV (3.7 Å, 5IZ7) [ 54 ], WNV (10 Å, 3J0B [ 55 ]/3.1 Å, 7KVA [ 56 ]) and chimeric DENV2/Binjari virus (2.5 Å, 7KV8 [ 56 ]).…”

“…A study on West Nile virus (WNV), another flavivirus, showed that a fusion loop binding antibody, E53, has a time- and temperature-dependent neutralization profile [ 68 ]. This finding suggests that the dynamic nature of E proteins is required for binding, despite its compact appearance of the E proteins on the virus surface under cryoEM [ 56 , 68 ]. Another cross-reactive neutralizing mouse antibody (MAb), 1A1D-2, recognizes an epitope on DIII with 18% of the epitope hidden on the smooth compact DENV2 surface [ 69 ].…”

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.…”

“…(B) Sequence alignment of stem regions of different flaviviruses using MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft, accessed on 5 July 2021) and ENDscript (http://espript.ibcp.fr/ESPript/ ESPript/, accessed on 5 July 2021) [19]). The yellow star above the sequences labels the conserved tryptophan targeted in this study, the blue dots below the contact residues of E in the pocket site 1 with lipids [12]. TBEV, tick-borne encephalitis virus; OHFV, Omsk hemorrhagic virus; POWV, Powassan virus; KSIV, Karshi virus; DENV1-4, dengue virus serotypes 1-4; ZIKV, Zika virus; SPOV, Spondweni virus; WNV, West Nile virus; JEV, Japanese encephalitis virus; NTAV, Ntaya virus; YFV, yellow fever virus; WSLV, Wesselsbron virus; YOKV, Yokose virus; ENTV, Entebbe bat virus; RBV, Rio Bravo virus; MODV, Modoc virus.…”

“…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).…”