Figure 1.
Summary of the Structural Organization and Different Conformations of the Flavivirus Envelope Protein E
(A) Schematic top view of the organization of the sE protein dimer as present at the surface of mature virions, color-coded according to the three domains (DI, DII, and DIII). The FP is indicated in orange.
(B) Crystal structure (top view) of the TBE virus sE dimer [31] represented as a ribbon diagram.
(C) Schematic side view of the E dimer at the surface of mature virions, with the “stem” and TM C-terminal polypeptide segments (missing in the truncated sE form) indicated in green. The viral lipid bilayer is illustrated with lipids belonging to the outer and inner leaflets colored blue and pink, respectively. Cryoelectron microscopy 3D reconstructions have shown that the stem forms two α-helices (H1 and H2) lying on the viral membrane, followed by the two TM segments [37].
(D) Schematic diagram illustrating the icosahedral arrangement of E dimers at the surface of mature flavivirus particles—in a “herringbone” pattern—as determined for dengue and West Nile virus [29,30]. Ninety E dimers form a rigid glycoprotein cage enclosing the viral membrane.
(E) Structure of the TBE virus sE in its trimeric postfusion conformation [33], represented as a ribbon diagram. Compared to the structure of E in the prefusion dimer, DIII is translocated (in a movement indicated by the light-blue arrow) to a lateral position, with its C terminus (labeled C-ter) projecting toward the FPs, thus generating a hairpin-like conformation.
(F) Schematic representation illustrating the proposed organization of full-length E in its postfusion conformation. In this model, the α-helices of the stem interact with the body of the trimer, in the grooves between adjacent, parallel DIIs. The lipid bilayer as well as the stem and TM segments are drawn as in (C).
(G) Top view of the sE trimer. For clarity, one of the subunits is colored according to domains, and the other two are given in a single color each (green and gray). The AB loop of DI (labeled in the figure [G]) rearranges upon dislocation of DIII, to make most of the DI–DI trimeric contacts. The relocated DIII acts as an external clamp, inserting into the grooves between DIs and providing additional intersubunit contacts. The vertical 3-fold axis at the center is indicated by a black triangle.
(H) Schematic drawing to simplify the top view of the sE trimer, matching the color coding of (G) (except for the subunit in yellow, which corresponds to the one colored by domains in [G]), to highlight the trimer-stabilizing role of DIII in the hairpin-like conformation of the molecule.
Figure 2.
Sedimentation Analysis Demonstrating the Alkaline pH–Induced Dissociation of E and Its Reversibility
(A) Virions were incubated for 10 min at pH 10.0 (boxes), pH 5.4 (diamonds), or pH 8.0 (circles), solubilized with Triton X-100, and analyzed at the respective pH by sedimentation in 7% to 20% sucrose gradients (w/w) containing 0.1% Triton X-100.
(B) Material from the alkaline pH–induced E monomer peak in Figure 2A was back-neutralized to pH 8.0 and then centrifuged at pH 8.0 into 7% to 20% sucrose gradients (w/w) in buffer containing 0.1% Triton X-100.
(C) Alkaline pH–treated virions were readjusted to pH 8.0, then solubilized with Triton X-100, and centrifuged at pH 8.0 in 7% to 20% sucrose gradients (w/w) in buffer containing 0.1% Triton X-100.
The sedimentation direction is from left to right, and the positions of E monomer (M), dimer (D), and trimer (T) are indicated.
Figure 3.
SDS-PAGE of TBE Virus Samples Cross-Linked with DMS at pH 8.0 and 10.0 without Pretreatment and after Pretreatment at pH 5.4
(A and B) SDS-PAGE of TBE virus samples cross-linked with DMS at pH 8.0 without pretreatment (A) and after pretreatment at pH 5.4 (B).
(C and D) SDS-PAGE of TBE virus samples cross-linked with DMS at pH 10.0 without pretreatment (C) and after pretreatment at pH 5.4 (D).
DMS concentrations are indicated on top of the individual lanes. Staining was performed with Coomassie blue. Positions of the E monomer (M), dimer (D), and trimer (T) are indicated.
Figure 4.
Electron Micrographs of TBE Virus at pH 8.0, 10.0, and 5.4
TBE virus was preincubated at pH 8.0 (A), 10.0 (B), and 5.4 (C), fixed with formalin, and negatively stained by phosphotungstic acid adjusted to pH 8.0 (samples A and B) or pH 6.0 (sample C). Arrows in (B) point to the rough surface generated by alkaline pH and in (C) to the bulky spikes generated by low pH treatment. All micrographs have been recorded at the same magnification. In (B) and (C), the virions lost their shell-like icosahedral envelope structure, at least at the particle surface, and as a consequence display irregular shapes that give the impression that the virus diameter is smaller than in (A). However, in all cases, the core diameter of the best-preserved virions has a similar value. In (C), the virions are aggregated, a characteristic of TBE virus maintained at low pH.
Figure 5.
Analysis of the Interaction of TBE Virus with Target Membranes by Liposome Coflotation
(A) Coflotation of virus with liposomes at alkaline pH. Virus was incubated for 10 min with liposomes at 37 °C at pH 10.0 (boxes) and as controls at pH 5.4 (diamonds) or pH 8.0 (circles), back-neutralized, and then subjected to centrifugation in sucrose step gradients. The gradients were fractionated, and the amount of E protein in each fraction was determined by a quantitative four-layer ELISA after denaturation of the samples with 0.4% SDS. The top fractions containing virus bound to liposomes are indicated by a bracket.
(B) Percentage of E protein bound to liposomes at different pHs compared with the control at pH 5.4.
(C) Percentage of E protein bound to liposomes at pH 10.0 after preincubation with different monoclonal antibodies compared with the control without monoclonal antibodies.
Figure 6.
Analysis of Membrane Fusion, E Dissociation, and Trimerization at Different pH Values
(A) Fusion of pyrene-labeled TBE virus with liposomes was carried out at 37 °C at the following conditions: (i) virus and liposomes were mixed an adjusted to pH 5.4, (ii) virus and liposomes were mixed and kept at pH 8.0, (iii) virus and liposomes were mixed and adjusted to pH 10.0, and (iv) virus and liposomes were preincubated at pH 10.0 for 10 min before adjustment to pH 5.4. The corresponding curves are labeled pH 5.4, pH 8.0, pH 10.0, and pH 10.0/5.4, respectively.
(B) Extent of low pH–induced fusion of pyrene-labeled TBE virus pretreated at pH 10.0 in the absence (control) and the presence of the FP-specific monoclonal antibody A1. The figure shows the values obtained 1 min after acidification.
(C) Sedimentation analysis demonstrating the low pH–induced trimer formation of virions preincubated at pH 10.0. Virions and liposomes were preincubated at pH 10.0 for 10 min, adjusted to pH 5.4 (filled circles), solubilized, and subjected to sucrose density centrifugation as described for Figure 2. As controls (dotted lines), virions were incubated for 10 min at pH 10.0 (boxes), pH 5.4 (diamonds), or pH 8.0 (open circles), solubilized, and analyzed as described above. The sedimentation direction is from left to right, and the positions of E monomer (M), dimer (D), and trimer (T) are indicated.
Figure 7.
Electron Micrographs of TBE Virus Interacting with Liposomes at pH 5.4 and 10.0
(A) Electron micrographs of a virus particle in the process of low pH–induced fusion with a liposome. Solid arrow points to low pH–induced projections at the virion surface; dotted arrow points to an electrodense structure presumed to be the nucleocapsid in the process of release.
(B) Virus particles attached to liposomal membranes at alkaline pH. Negative stain by phosphotungstic acid adjusted to pH 8.0.