Membrane curvature in flaviviruses

Abstract

Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by the icosahedral scaffold composed of envelope (E) and membrane (M) proteins through interaction of the proteins' stem-anchor regions with the membrane. In localized regions, small membrane areas exhibit convex, concave, flat or saddle-shaped surfaces that are constrained by the specific protein organization within each membrane leaflet. These results suggest that the organization of membrane proteins in small enveloped viruses mediate the formation of membrane curvature.

Keywords: 3D; Cryo-electron microscopy; DENV; Dengue virus; Enveloped virus; Flavivirus; Membrane curvature; WNV; West Nile virus; cryo-EM; cryo-electron microscopy; dengue virus; three-dimensional.

Fig. 1

Cryo-EM map of mature WNV at 10.3 Å resolution. (A) The reconstruction map showing the external protein shell in blue and the N-linked carbohydrates in red. The numbers mark the positions of the icosahedral two, three- and five-fold axes. (B) The fitted E dimers arranged as a herringbone-like raft structure on the surface of WNV. The icosahedral two-fold dimer is in orange, the two other quasi-related E molecules in the same asymmetric unit are colored in blue and green. The fusion peptide is colored in red. The line “p” represents the position of the cross section in C. (C) Cross section of the cryo-EM density map. The lines labeled “a” (icosahedral two-fold), “b” and “c” indicate the directions of the corresponding radial density plots displayed in panel C. (D) Radial density plots along “a”, “b” and “c” in C. The green bar represents the peak-to-peak distance between the centers of the inner and outer membrane leaflets.

Fig. 2

Diagrams illustrating the calculation of membrane curvature and thickness. (A) Six elementary triangles (f₁ to f₆) that approximate a membrane surface at point P. O represents the center of the virus particle. (B) Side view of two neighboring triangles that approximate a membrane surface. (C) Diagram showing the calculation of membrane thickness. S_out and S_in are surfaces symbolizing the outer and inner membrane leaflets. S_mid is the surface representing the lowest density region between the two leaflets. O is the center of the virus particle in the reconstruction map.

Fig. 3

Positions of and distance between the centers of the inner and outer membrane leaflets in WNV. (A) Cut-away view showing the E protein shell (blue), membrane bilayer (green) and the nucleocapsid core (purple). (B–C) Radial projections of the WNV cryo-EM map at a radius corresponding to the stem (B) and the transmembrane helices of the E and M proteins (C), respectively. (D) Radial position of the inner membrane leaflet color-shaded (blue) and contoured at 3.0 Å intervals. The blue contour lines closest to the five-fold axes demarcate positions of 159 Å radii. The highest electron densities, corresponding to the transmembrane helices of the E and M proteins in the inner membrane leaflet, are contoured (red lines) at 0.3σ intervals and 2.1σ above the background of the cryo-EM map. (E) Radial position of the outer leaflet color-shaded (blue) and contoured at 3.0-Å intervals. The blue contour lines closest to the five-fold axes demarcate positions of 188 Å radii. The electron densities corresponding to the stem regions of E and M proteins in the outer membrane leaflet are contoured (red lines) at 0.3σ intervals and 1.9σ above the background of the reconstruction map. (F) Distance between the centers of the inner and outer membrane leaflets in WNV. The green contour lines are displayed at 2.0 Å intervals with the one closest to the two-fold axis representing 22.6 Å. The red contour lines show the electron densities of the E and M transmembrane helices at a position midway between the outer and inner membrane leaflet. These densities were contoured at 0.3σ level intervals and 1.3σ above the background in the reconstruction map.

Fig. 4

Membrane curvature in mature WNV particles. (A–B) The average curvatures of the inner (A) and outer (B) membrane leaflets superimposed on the red contour lines that represent the protein densities at the respective membrane leaflet. The positive curvatures (blue), represent the convex areas viewing from outside of the particle. The negative curvatures (red) represent the concave areas. (C–D) The Gaussian curvatures of the inner (C) and outer (D) membrane leaflets. The negative Gaussian curvatures (red) correspond to local saddle-shaped surfaces. “a” represents the direction either pointing to two neighboring rafts (C), or circling the five-fold axis of the membrane (D). “b” represents the direction perpendicular to “a”. (E) A saddle-shaped membrane formed in a region where proteins (blue and orange) bend the membrane away from its natural curvature. “a” and “b” represent two bending directions of the membrane depicted as dark green and purple (applicable to F). (F) A saddle-shaped membrane area (dark green circles) located at the edge of an invaginating membrane area surrounded by a group of proteins (blue).

Fig. 5

Membrane position and curvature in the immature DENV particle. (A) Cryo-EM reconstruction of an immature DENV particle at 16 Å resolution (Zhang et al., 2003b) with the outer membrane leaflet colored in green and the protein shell in orange. The line “P” represents the position of the cross-section shown in panel B. (B) Cross-section of the immature dengue virus cryo-EM density. “q3” represents the center of the protein spike which has a quasi-threefold symmetry. The arrows point to the protein spikes that cover the membrane areas having distinctly smaller radii. The scale bar is 100 Å. (C) Radial projection of the immature DENV cryo-EM map at a radius of 180 Å, which corresponds to the center of the viral membrane. The dark densities correspond to the transmembrane domains of E and M proteins. (D) Radial position of the inner leaflet shaded (blue) and contoured at 3 Å intervals. The blue contour lines closest to the q3 axes demarcate the positions of 164 Å radii. The red contour lines represent the electron densities of the E and M transmembrane helices at the inner membrane leaflet. These densities are contoured at 0.3σ interval and are 2.0σ above the background level of the reconstruction map. (E) Radial position of the outer leaflet shaded (blue) and contoured at 3 Å intervals. The blue contour lines closest to the q3 axes demarcate the positions of 191 Å radii. The electron densities of the E and M proteins at the outer membrane leaflet are contoured (red lines) at 0.3σ intervals and 2.2σ above the background of the reconstruction map. (F–G) The average curvatures of the inner and outer membrane leaflets superimposed on the contours of the highest electron densities within the respective leaflet.