Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

Abstract

Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur.

Importance: Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments.

FIG 1

Structure of the E1E2 heterodimer of chikungunya virus (PDB 3N42). The amino and carboxy E2 β-ribbon connectors to domain B are colored brown, and the E1 fusion loop is colored black. Red and blue spheres represent the amino acids in the Fab 8B10 and 5F10 footprints, respectively. Gray spheres represent escape mutants.

FIG 2

Cryo-EM reconstructions of CHIK-VLP in complex with Fab fragments of neutralizing antibodies looking down a 2-fold axis. One icosahedral asymmetric unit is outlined in white. The surface of the complex is colored according to its distance from the center of the virus as given by the inset scale in angstroms. Thus, the Fab fragment bound to the surface of the virus is blue, since it is at a greater distance from the center of the virus.

FIG 3

Fit of the Fab structures (yellow) into the cryo-EM density difference maps (blue mesh) that were calculated by performing a complete three-dimensional orientational search and a limited three-dimensional positional search using the program EMfit. The E1 glycoprotein is colored in red, and E2 is colored cyan.

FIG 4

Stereographic projection showing a roadmap of CHIK-VLP with Fab 8B10 binding sites. The right-hand panel is a close-up of the leftmost trimeric glycoprotein spike. The amino acid colors represent the distance from the center of the virus as defined by the color scale bar. Domains A and B and the β-ribbon connector are bounded by black, white, and dashed yellow lines, respectively. Blue dashes represent the boundaries of the putative receptor-binding region. White-shaded residues represent where the Fab binds on E2. Positions on the surface of the virus can be identified by their latitude and longitude as is usual for locations on Earth. The edges of the left-hand figure correspond to lines of latitude and longitude.

FIG 5

Stereographic projection showing a roadmap of CHIK-VLP with Fab 5F10 binding sites. The right-hand panel is a close-up of the leftmost trimeric glycoprotein spike. The amino acid colors represent the distance from the center of the virus as defined by the color scale bar. Domains A and B and the β-ribbon connector are bounded by black, white, and dashed yellow lines, respectively. Blue dashes represent the boundaries of the putative receptor-binding region. White-shaded residues represent where the Fab binds on E2.

FIG 6

Overview of a single trimeric spike showing positions of residue Arg82 (red spheres). Each E1E2 heterodimer is colored separately for clarity.