Immature and mature dengue serotype 1 virus structures provide insight into the maturation process

Abstract

Dengue virus is a major human pathogen that has four serotypes (DENV1 to -4). Here we report the cryoelectron microscopy (cryo-EM) structures of immature and mature DENV1 at 6- and 4.5-Å resolution, respectively. The subnanometer-resolution maps allow accurate placement of all of the surface proteins. Although the immature and mature viruses showed vastly different surface protein organizations, the envelope protein transmembrane (E-TM) regions remain in similar positions. The pivotal role of the E-TM regions leads to the identification of the start and end positions of all surface proteins during maturation.

Fig 1

Cryo-EM structures of the DENV1. (A) Cryo-EM density of immature virus. The scale bar is 100 Å. The density is colored radially: radii between 0 and 160 Å, between 161 and 210 Å, between 211 and 270 Å, and 271 Å and above are colored in yellow, green, light blue, and magenta, respectively. For clarity, the cryo-EM maps in panels A and B were low-pass filtered to an 8-Å resolution. (B) Cryo-EM density of mature DENV1. The density is colored radially: radii between 0 to 160 Å, between 161 and 210 Å, between 211 and 250 Å, and 251Å and above are colored in yellow, green, light blue, and magenta, respectively. (C and D) Zoomed-in view of fitted surface proteins into the cryo-EM density maps (gray) of immature (C) and mature (D) virus. Black arrows in panel D point to density corresponding to glycosylation sites. DI, DII, and DIII of the E ectodomain are colored in red, yellow, and blue, respectively. The pr part of the prM molecule is colored in cyan, and the M protein is colored in purple. The E stem and transmembrane (TM) regions are colored in dark green. (E) E protein stem helix α2 fitted into the 4.5-Å-resolution mature DENV1 virus map. Densities of some side chains are resolved (black arrows). The side chain models were not determined experimentally and are shown to indicate the protein sequence. (F) Close-up view of a part of the E protein DI main chain in the 4.5-Å-resolution map of the mature DENV1 virus. The densities for β-strands are separated. (G) Fourier shell correlation versus resolution plots for the individual lipid bilayer (red) and the E-prM protein shell (green) of the immature DENV1 map and the shell including the lipid bilayer and the outer surface protein of the mature DENV1 (blue).

Fig 2

Organization of the E and prM ectodomains in the immature DENV1 and their interactions. (A) Organization of the E and prM protein trimeric spikes on the surface of the virus. Three molecules in the asymmetric unit are shown as ribbons and are labeled A, B, and C. Symmetry-related molecules are shown as surfaces, and E protein surfaces are colored in gray whereas the pr surfaces are colored in cyan. The symmetry-related molecules are labeled a, b, and c. The black triangle represents one asymmetric unit. (B) Structure of an E protein complexed with prM protein. Color schemes are defined in Fig. 1. (C) Interactions between pr molecules (cyan) at the tip of the spike. Residues are identified as interacting by having a distance of less than 8 Å between Cα chains. (C to F) The interacting residues are colored in orange, and the interacting interface is indicated by an asterisk. (D) Stereodiagram showing the interactions between DII (yellow) of the molecules (labeled A, B, and C) in the asymmetric unit and DIII (blue) from neighboring symmetry-related molecules (labeled a, b, and c). (E) Interactions between M molecule (pink) and the DII (yellow) of the E ectodomain. The furin cleavage site on the prM protein is colored in green and marked with an “X.” (F) Interactions of the E stem region (dark green) with DI of the E protein (red). (G) Fit of the E protein stem α-helices into the 6-Å cryo-EM map. The density that connects the DIII of the E protein to the stem region (indicated by “*”) and the stem to the transmembrane region (indicated by “#”) can be observed.

Fig 3

Organization of ectodomain E on the mature DENV1 and their interactions. (A) Organization of ectodomain E on the surface of DENV1. DI, DII, and DIII are colored in red, yellow, and blue, respectively. The black triangle represents an asymmetric unit. (B) Interactions between the molecules within the E ectodomain dimers. A raft containing two asymmetric units is shown here. (B to F) Interacting residues are colored in green. (C) Interdimeric interactions between the E ectodomains in the raft. (D) Interactions between DIIIs (blue) near the 5-fold vertex. (E) Interactions between E ectodomains near the 3-fold vertex. (F) Interactions between two E ectodomain rafts that form the herringbone pattern on the virus surface. (G) Binding site of the pr molecule on the E protein (6), shown in mature virus. Residues bound by the pr molecule are highlighted in green.

Fig 4

Organization of membrane-associated proteins in the mature DENV1 virus. (A) Organization of the stem and TM regions of E proteins (dark green) and M proteins (purple) in the virus. The black triangle represents an asymmetric unit. The M protein forms homodimeric interactions. (B) M ectodomain (N-terminal residues 1 to 26) and stem region (N-terminal residues 27 to 39) (black rectangle box) in the mature DENV. Domains DI and DII of the interacting E protein are shown in red and yellow, respectively. (C) Stereodiagram of the interactions of the M protein (purple) with the other molecule (pink) in the homodimeric structure. (C to E) Interacting residues are colored in light green. (D) Stereodiagram of the interactions of the M protein with the stem and TM regions of the E protein. (E) Open-book view of the interactions between the E ectodomain and the M protein as well as the stem and TM regions of E protein. View of the E ectodomain from inside the virion while the TM regions are viewed from the outside.

Fig 5

Comparing the surface E proteins between the mature DENV1 and DENV2. (A) Electrostatic charges on the surfaces of DENV1 and DENV2. Surfaces of a raft consisting of two asymmetric units of the E ectodomain are shown. Positive and negative charges are colored in blue and red, respectively. The DENV2 surface contains larger numbers of positively charged residues. (B) Location of nonconserved residues on an E ectodomain raft. The nonconserved residues (spheres) are present on all domains of the E ectodomain protein except at the hinge between DI and DII, the hinge between DI and DIII, and the fusion loop at the tip of DII. (C) Location of nonconserved residues on an E ectodomain, side view. The epitope recognized by highly potent serotype-specific antibodies is located at the lateral side of DIII (purple circle). The epitopes bound by weakly neutralizing antibodies, which are generally cross-reactive to all serotypes, are circled in green.

Fig 6

Movement of surface proteins during DENV maturation. (A) Movement of the E stem region during maturation. The positions of TM1 and TM2 of the E proteins of the immature and mature virus are aligned so as to compare the positions of the stem α1 and α2. Helix α1 is rotated by about 180 degrees relative to α2, and α2 is rotated approximately 45 degrees counterclockwise relative to TM1. (B) Positions of the stem and TM regions of the E protein in immature (left) and mature (right) structures. There are some localized rotations occurring in the stem region and also the TM region of the E proteins during maturation. However, the position of the E transmembrane in the asymmetric unit of the virus did not change significantly between the immature and mature virus. (C) Rearrangement of the E ectodomain molecules in the immature and mature structures. The three independent E protein molecules in the asymmetric unit are colored red, green, and blue. The positions of their TM regions allow us to identify which position the E molecule in the immature virus structure moves into in the mature virus structure—red moves to red, green moves to green, and blue moves to blue. (D) Movements of the M protein. The three independent M protein molecules in the asymmetric unit are colored in red, green, and blue. There is significant movement of the M proteins in the virus membrane.