Structural studies of Chikungunya virus maturation

Abstract

Cleavage of the alphavirus precursor glycoprotein p62 into the E2 and E3 glycoproteins before assembly with the nucleocapsid is the key to producing fusion-competent mature spikes on alphaviruses. Here we present a cryo-EM, 6.8-Å resolution structure of an "immature" Chikungunya virus in which the cleavage site has been mutated to inhibit proteolysis. The spikes in the immature virus have a larger radius and are less compact than in the mature virus. Furthermore, domains B on the E2 glycoproteins have less freedom of movement in the immature virus, keeping the fusion loops protected under domain B. In addition, the nucleocapsid of the immature virus is more compact than in the mature virus, protecting a conserved ribosome-binding site in the capsid protein from exposure. These differences suggest that the posttranslational processing of the spikes and nucleocapsid is necessary to produce infectious virus.

Keywords: Chikungunya virus; alphavirus; conformational changes; cryo-electron microscopy; maturation.

Fig. 1.

Cryo-EM reconstruction of immature CHIK virus-like particle. (A) Three-dimensional cryo-EM map of immature CHIKV, viewed down an icosahedral twofold axis. An icosahedral asymmetric unit is marked by a black triangle. Icosahedral symmetry elements are shown as black-filled pentagon, triangles, and ellipse. Four unique subunits in an asymmetric unit are shown in white numbers. (B) Internal capsid protein shell of the immature CHIKV. (C) Central cross-sections of the immature CHIKV viewed down an icosahedral twofold axis. Components of the virus are shown in different colors as indicated in the figure. (D) Enlarged view of the region outlined by the black rectangle in C. Fitting of an E1-p62-C structure is shown in the cryo-EM map of immature CHIKV.

Fig. 2.

Structural characteristics of the immature CHIKV. (A) A trimeric spike of the immature CHIKV. The E2 molecules (red) form interactions within a spike whereas the E1 molecules (yellow) wrap around E2 molecules and form interactions between spikes. The E3 molecules (green) are located at the periphery of the E2 molecules. (B) Central cross-section of the immature (Left) and mature (Right) CHIKV. The icosahedral symmetry axes are indicated by white arrows. Components of the viruses are shown in different colors as indicated in the figure. The immature virus has smaller holes around the i2 and i5 symmetry axes compared to the mature virus. The diameter of the nucleocapsid in the immature virus is smaller than in the mature virus.

Fig. 3.

Comparisons of the E1-E2-C structure in the immature and mature CHIKV. (A) Superposition of the E1-p62-C structure in the immature conformation (magenta) to E1-E2-C structure in the mature conformation (cyan). The E3 molecule in p62 is in blue. The capsid protein has a similar orientation in both immature and mature conformations. However, the TM helices have a different orientation and location in the immature form compared with the mature form. (B) Density of the capsid protein. The ordered structure of the capsid protein consists of residues 113–261. Additional density seen only in the cryo-EM reconstruction of the virus (outlined with black dashes) belongs to the N-terminal region of the capsid protein. This region is the RBS (residues 98–112). The hydrophobic pocket of the capsid protein is indicated by a red star.

Fig. 4.

Gold-standard FSC curve for refinement. The resolution corresponding to the 0.143 FSC cutoff is 6.8 Å.