Structure of tick-borne encephalitis virus and its neutralization by a monoclonal antibody

Abstract

Tick-borne encephalitis virus (TBEV) causes 13,000 cases of human meningitis and encephalitis annually. However, the structure of the TBEV virion and its interactions with antibodies are unknown. Here, we present cryo-EM structures of the native TBEV virion and its complex with Fab fragments of neutralizing antibody 19/1786. Flavivirus genome delivery depends on membrane fusion that is triggered at low pH. The virion structure indicates that the repulsive interactions of histidine side chains, which become protonated at low pH, may contribute to the disruption of heterotetramers of the TBEV envelope and membrane proteins and induce detachment of the envelope protein ectodomains from the virus membrane. The Fab fragments bind to 120 out of the 180 envelope glycoproteins of the TBEV virion. Unlike most of the previously studied flavivirus-neutralizing antibodies, the Fab fragments do not lock the E-proteins in the native-like arrangement, but interfere with the process of virus-induced membrane fusion.

Fig. 1

Structure of TBEV virion. a Cryo-EM image of TBEV virions. The sample contained mature, immature (white arrows), half-mature (white arrowheads), and damaged (black arrows) particles. Scale bar 100 nm. b B-factor sharpened electron-density map of TBEV virion, rainbow-colored according to distance from particle center. The front lower-right eighth of the particle was removed to show the transmembrane helices of E-proteins and M-proteins. c Molecular surface of TBEV virion low-pass filtered to 7 Å. The three E-protein subunits within each icosahedral asymmetric unit are shown in red, green, and blue. The three E-proteins in the icosahedral asymmetric unit form unique interactions with each other (for more details, see Supplementary Fig. 2). The black triangle shows the borders of a selected icosahedral asymmetric unit. d Central slice of TBEV electron density map perpendicular to the virus 5-fold axis. The virus membrane is deformed by the transmembrane helices of E-proteins and M-proteins. The lower right quadrant of the slice is color-coded as follows: nucleocapsid—blue; inner and outer membrane leaflets—orange; M-proteins—red; E-proteins—green. Scale bars in b, c, and d represent 10 nm

Fig. 2

Structure and organization of the E-proteins and M-proteins in TBEV virion. a Dimer of E-proteins with domain I colored in red, domain II in yellow, domain III in violet, and domain IV in blue. The electron density map of one of the proteins is shown as a semi-transparent surface. Glycosylation site Asn157 and residues Trp101 and Phe108 from the fusion loop of domain II are shown in detail. b Heterotetramer of two E-proteins and two M-proteins. E-proteins are colored according to domains, and M-proteins are shown in orange. c Superposition of cryo-EM (colored) and X-ray (gray) E-protein structures. The cryo-EM structure includes three perimembrane (h1–h3) and two transmembrane helices (h4 and h5). d M-protein rainbow-colored from N-terminus in blue to C-terminus in red with electron density map shown as semi-transparent surface. The M-protein consists of an extended N-terminal loop followed by perimembrane (h1) and two transmembrane helices (h2 and h3). e Molecular surface of E–M heterotetramer. Histidines with a putative role in the dissociation of the heterotetramers are shown in magenta. Insets show details of interactions of the histidine side chains. f Structures of E-proteins and M-proteins colored according to conservation of amino acid sequence among viruses from the family Flaviviridae (for more details, see Supplementary Fig. 5). Insets show the conservation of histidines that might be involved in the pH-dependent dissociation of the heterotetramers. g Molecular surface of E–M heterotetramer colored according to electrostatic potential at pH 8.5 and 5.8. One E–M heterodimer is shaded for clarity. Insets show the surface potential surrounding the histidines that might be involved in the pH-dependent dissociation of the hetrotetramer. The table in the middle lists the pKa values of the selected histidines calculated using Rosetta-pKa

Fig. 3

Neutralization of TBEV by antibody 19/1786. Dose-dependent neutralization curves of TBEV treated with IgG 19/1786 (a) and Fab 19/1786 (b). Error bars represent standard deviation of the measurements (IgG n = 5; Fab n = 2). EC₅₀ values with standard error of the mean are shown in the graph. c Dose-dependent neutralization curves of TBEV and its mutants treated with IgG 19/1786. Mutations in domain I (red circle—double mutant; blue triangle—quadruple mutant) and domain II (magenta triangle—double mutant; green diamond—quadruple mutant) affect the neutralization activity of Fab 19/1786 compared to the wild-type virus (black square). The mutations in the domains are listed in the top left corner. The error bars represent the standard deviation of the measurements (n = 3). Calculated EC₅₀ values (µg ml⁻¹) with standard errors are listed in the bottom right corner

Fig. 4

Interaction of TBEV virions with Fab fragments of neutralizing antibody 19/1786. a Cryo-EM micrograph of TBEV virions incubated with Fab fragments of 19/1786. Scale bar represents 100 nm. b Electron-density map of Fab-covered TBEV virion rainbow-colored according to distance from center of particle. The right half of the image represents a B-factor sharpened map. Electron densities corresponding to the Fab fragments are located close to the 3-fold and 5-fold symmetry axes of the virion. c Molecular surface of TBEV virion covered with Fab 19/1786 fragments low-pass filtered to 7 Å resolution. E-proteins are shown in red, green, and blue. Fab fragments are shown in magenta (heavy chain) and pink (light chain). Scale bars in b and c represent 10 nm. d Footprints of Fab 19/1786 on TBEV surface. Fab fragments bind to two of the E-proteins of the asymmetric unit. The Fabs interact with an additional E-protein from the neighboring asymmetric unit shown in faint colors. e The Fab 19/1786 binds to the domain III at an angle of 135° relative to the axis of the E-protein ectodomain. The E-protein domain I is shown in red, domain II in yellow, domain III in violet, and domain IV in blue. The heavy chain of the Fab fragment is shown in magenta and the light chain in pink. The leaflets of the viral membrane are represented by gray dashed lines

Fig. 5

TBEV virions and TBEV–Fab 19/1786 complex in low-pH environment. a Cryo-EM micrographs of TBEV virions fused together under low-pH conditions. However, TBEV virions covered with Fab 19/1786 fragments did not aggregate. Scale bar represents 100 nm. b “Fusion-from-without” assay on C6/36 cells. The control cells did not fuse in the low-pH (5.5) environment. In contrast, native TBEV induced complete cell fusion. Pre-incubation of TBEV with IgG 19/1786 completely abolished the fusion activity of the virus and the Fab 19/1786 lowered the fusion activity of the TBEV. The scale bar represents 100 µm. c Cryo-EM reconstruction of Fab-covered TBEV virion at low pH. The isosurface is radially colored according to the distance from the center. Corresponding central slice perpendicular to the 5-fold axis is shown on the right. The particles lost the compact layer of the E-protein ectodomain and the viral membrane is not deformed by the transmembrane helices. The lower right quadrant of the slice is color-coded as follows: nucleocapsid—blue; inner and outer membrane leaflets—orange; M-proteins, E-proteins, and Fab fragments cannot be distinguished and are shown in green. The scale bar represents 25 nm. d Reference-free 2D class averages of TBEV virions in solutions with different pH levels. The upper half of the images shows the class averages, whereas the bottom parts are radial averages of the same classes. In the radial averages, the three distinct layers represent the inner and outer leaflets of the membrane (in orange) and the ectodomain of the E-proteins (in green). An additional double layer is visible on the virion covered with Fab fragments (in cyan). The layer corresponding to ectodomains of E-proteins and Fab fragments is more diffuse in the low-pH structure than in the particles at neutral pH. The viral membrane remained fully preserved, but lost the deformities introduced by the transmembrane helices of virus proteins. The 2D class averages are color-coded as follows: nucleocapsid—blue; inner and outer membrane leaflets—orange; E-proteins—green; Fab 19/1786 attached to virus surface—cyan. The scale bar represents 25 nm