Structure of dengue virus: implications for flavivirus organization, maturation, and fusion

Abstract

The first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal beta barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

Figure 1. The CryoEM Density

(A) Surface-shaded representation of dengue-2 cryoEM reconstruction at 24 Å resolution, showing the outline of one icosahedral asymmetric unit and the definition of the coordinate system. Scale bar represents 100 Å. (B) Central crosssection showing the cryo-EM density with a plot of the maximum (blue) and averaged (purple) density. Arrows indicate the position of the 5-fold and 3-fold axes. Shown also are radial density sections at the defined radii, r1, r2, r3, and r4. Higher density representing protein is shown in dark shading. Scale bar represents 175 Å. (C) Ribbon drawing of the E dimer situated on an icosahedral 2-fold axis, showing the largest uninterpreted electron density peak outside the lipid bilayer, probably representing the M protein (light blue), located close to the hole between the E dimers. The white arrow indicates the position of the dimer holes. The outer leaflet of the lipid bilayer is shown in green. The domains I, II, and III of an E monomer are shown in red, yellow, and blue, respectively. The fusion peptides are in green. (D) Ribbon drawing showing the position and orientation of the E dimer associated with an icosahedral 2-fold axis. Shown in white is the outline of one icosahedral asymmetric unit. The domains of E are colored as in (C). The portion of the membrane protein M below the dimer is indicated.

Figure 2. CryoEM Density of the Nucleocapsid Shell and the RNA

(A) Stereo diagram of the region corresponding to the yellow nucleocapsid shell in Figure 1, between 105 and 135 Å radii. (B) Stereo diagram of density corresponding to the RNA region of the core (red in Figure 1), inside a radius of 105 Å. Only one hemisphere is shown. Scale bars represent 100 Å.

Figure 3. Fit of E Dimers into Density

(A) and (B) correspond to density that is between planes, perpendicular to an icosahedral 2-fold axis and at a distance of 220-250 A from the viral center. The contour level is at about 4σ (root mean square deviation from the mean density). (A) Stereo diagram showing only the density between 220 and 245 A radius. (B) Stereo diagram showing the interpreted density in terms of dimers on icosahedral 2-fold axes (green) and dimers on quasi-2-fold axes (red). (C) Structure of the whole virus showing each monomer with domains I, II, and III in red, yellow, and blue, respectively. The fusion peptide is shown in green. The C-terminal residue 395 is shown as a white asterisk for monomers within the defined icosahedral asymmetric unit. Note the pair of holes in each dimer. Scale bar represents 100 A. (D) Central crosssection through the cryo-EM density showing the outer radial shells as in Figure 1B. The direction of the crosssection follows the length of a dimer situated on an icosahedral 2-fold axis. The arrow indicates the position of the 2-fold axis. The location of domain III and the fusion peptide in domain II are shown. Asterisks indicate the carboxy end of the fitted E protein.

Figure 4. Proposed Rearrangement of E Dimers in Flaviviruses upon Exposure to Low pH

The E protein dimers in mature virus (A) are shown undergoing a rearrangement to the predicted T = 3 fusogenic structure (C) with a possible intermediate (B). Arrows in (B) indicate the direction of E rotation. The solid triangle in (C) indicates the position of a quasi-3-fold axis. Note the radial expansion of the particle in (B) and (C).

Figure 5. Configuration of Glycoproteins of Alphaviruses and Flaviviruses on the Surface of Virions at Neutral pH and the Proposed Configuration at Acid pH

(A) In the alphaviruses, E1 glycoproteins are shown as green cylinders, E2 glycoproteins as tan shapes, and the fusion peptide (FP) as a black curved line. (B) In flaviviruses, E glycoproteins are shown as yellow cylinders with the fusion peptide as a green curve. In both (A) and (B), the membrane is shown in gray.