Structural changes in dengue virus when exposed to a temperature of 37°C

Abstract

Previous binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed.

Fig 1

The DENV2 NGC strain undergoes structural changes after incubation at 37°C. The control (left) consists of DENV2 grown in a mosquito cell line at 28°C and then kept at 4°C during purification. The virus particles after incubation at 37°C change their morphology from a smooth surface (left) to a rough surface (right).

Fig 2

Three different classes of DENV2 NGC particles obtained with EMAN2. (A) Class II particles that have a rough surface. (B) Class III particles that have a relatively smooth surface. (C) Class IV particles that are smaller than the other classes.

Fig 3

Cryo-EM maps reconstructed from class I to IV particles present in the DENV2 sample incubated at 37°C. The surfaces of the maps (above) and their central cross-sections (below) are colored according to their radii (shown in the lower panel). The white triangle represents an icosahedral asymmetric unit, and the corresponding 2-, 3-, and 5-fold symmetry vertices are indicated. Maps generated from the class I particles are similar to the previous published DENV-2 maps (–9, 15). Class II and III maps showed that the particles in these classes have bigger radii, indicating that the virus had expanded. There are protruding densities on the virus surface between the 5- and 3-fold vertices (white arrows). The class IV map showed very poor density on the E protein layer, indicating that the E protein had lost its icosahedral symmetry.

Fig 4

The fit of E proteins into cryo-EM maps of class I and III particles. The maps are colored in light gray, whereas the fitted E protein structures are drawn in a wire representation and colored in red, yellow, and blue for domains I, II, and III, respectively. The icosahedral asymmetric unit of both maps is depicted as a black triangle with the corresponding symmetry axis indicated by numbers. The class I map (left) is similar to the previously published DENV-2 cryo-EM structure (–9, 15). The class III map (right) showed the presence of a hole at each of the 3-fold vertices. Also, domains I and III of the E protein molecule at the icosahedral 2-fold vertices are observed to be protruding (indicated by a black arrow).

Fig 5

Structural differences between class I and class III particles. (A) The densities of the cryo-EM maps of classes I and III are shown in mesh and colored according to their radii as shown in Fig. 3. The 15-Å gap between lipid bilayer and E proteins layer in class III particles is indicated. (B) Class I (left) and III (center) E protein rafts, each consisting of two asymmetric units, are shown. The three individual E proteins in the asymmetric unit are shaded and labeled A, B, and C and the same E proteins in the neighboring asymmetric unit are labeled A′, B′, and C′ (left). In the right panel, the two structures are superimposed, colored in cyan and orange for classes I and III, respectively. The E protein dimer (molecules A and C) near the 5-fold vertex in the class III structure rotates about 7° compared to the class I structure. The icosahedral 2-fold E protein dimers (molecules B and B′) in the class I structure have moved apart from each other in the class III structure. (C) Side view of the superimposed class I and III structures of the dimer consisting of molecules A and C (left) and molecule B (right). Molecule B of the class III structure rotates 14° clockwise from the class I structure using the fusion loop (black dot) as a pivot point.

Fig 6

Comparison of the class III structure with the Fab 1A1D-2–DENV complex structure. (A) Organization of the three individual E proteins in an icosahedral asymmetric unit of the class III structure (left) and Fab 1A1D-2–DENV complex structure (right). Domains I, II, and III are colored in red, yellow, and blue, respectively. (B) Differences in the orientations of molecule B in the class I, III, and Fab 1A1D-2–DENV complex structures. In the Fab 1A1D-2–DENV structure, molecule B is not bound by antibody, while the other E protein molecules are bound. The E protein is drawn as a surface representation. The antibody 1A1D-2 epitope is indicated in pink on the E protein surface. Conformational changes from class I to class III to the one observed in Fab 1A1D-2–DENV structures are shown.

Fig 7

Comparison of the accessibility of the 1A1D-2 epitope on the three individual E proteins in the asymmetric unit of class I and class III structures. Molecules A, B, and C (indicated in Fig. 5B) are colored in yellow, cyan, and green, respectively. The other neighboring E proteins are shown as a transparent white surface. The antibody (Ab) 1A1D-2 epitope is colored in red.

Fig 8

Comparison of the accessibility of the E111 epitope on the three individual E proteins in the asymmetric unit of class I and class III structures. The coloring scheme is the same as in Fig. 7. The 3-fold symmetry axis is indicated as a dashed line with a 3-fold symmetry (sym.) symbol. Holes on the class III surface are located at all 3-fold vertices.