Abstract
The current Zika virus (ZIKV) epidemic is characterized by severe pathogenicity in both children and adults. Sequence changes in ZIKV since its first isolation are apparent when pre-epidemic strains are compared with those causing the current epidemic. However, the residues that are responsible for ZIKV pathogenicity are largely unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of the immature ZIKV at 9-Å resolution. The cryo-EM map was fitted with the crystal structures of the precursor membrane and envelope glycoproteins and was shown to be similar to the structures of other known immature flaviviruses. However, the immature ZIKV contains a partially ordered capsid protein shell that is less prominent in other immature flaviviruses. Furthermore, six amino acids near the interface between pr domains at the top of the spikes were found to be different between the pre-epidemic and epidemic ZIKV, possibly influencing the composition and structure of the resulting viruses.
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Acknowledgements
We thank M. Sevvana for discussions about the manuscript. We thank S. Kelly for helping us prepare the manuscript. We also thank the Purdue Cryo-EM Facility for equipment access and support. This work was supported by the National Institutes of Health (RO1 AI076331 to M.G.R. and R.J.K., and a subaward for RO1 AI073755 (principal investigator: M.S. Diamond, Washington University) to both M.G.R. and R.J.K.).
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A.S.M., D.S. and G.B. were involved in preparation of cell culture, and optimization and purification of virus sample; V.M.P. and T.K. conducted the cryo-EM preparation, data collection and data processing; V.M.P. performed the data analyses; W.J. made his JSPR program available for reconstruction and refinement of the cryo-EM map; and V.M.P., R.J.K. and M.G.R. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Resolution estimation using Fourier shell correlation (FSC).
A plot of FSC against resolution in Å-1, calculated using two independent half-sets of the cryo-EM data. The resolution of the map is 9.1 Å using the 0.143 FSC cut-off (light blue line).
Supplementary Figure 2 Fit of the DENV-2 prM–E crystal structure into the immature ZIKV cryo-EM map.
a, Surface view of the complete immature ZIKV. One of the 60 trimeric spikes is identified in color, with its E proteins in green and the pr domains in orange. The asymmetric unit is shown as a black triangle. The scale bar is 100 Å long. b, Top view showing the fit of three prM-E heterodimers into the cryo-EM spike density. The three independent prM-E heterodimers are colored in light green, blue and orange for the E proteins and dark green, dark blue and red for the pr domains, respectively. c, Side-view of the fitted prM-E heterodimers in a spike. The dashed black line shows the position of the viral membrane surface. d, Cryo-EM density showing the individual fitting of the transmembrane regions of E (blue) and M (magenta) proteins. Scale bar is 50 Å long in b, c and d.
Supplementary Figure 3 Analysis of crystal structures fitted into immature ZIKV cryo-EM map.
a, Cryo-EM density showing the conservation of Asn-69 glycosylation site in the pr domains of immature ZIKV. The DENV-2 prM-E crystal structure (PDB ID: 3C6E) including the Asn-69 glycan has been fitted into all three positions within a trimeric spike. The glycans attached to Asn-69 are shown in blue. Scale bar is 25 Å in length. b, Glycan (colored in blue) attached to Asn-67 in the DENV-2 E protein structure and fitted into the map of the immature ZIKV showing the absence of density associated with the glycan at this site in ZIKV. c, Additional density adjacent to Asn-154 (red) in immature ZIKV indicating the presence of a glycan. Scale bar is 10 Å long in b and c. Black arrows in a and c point to the densities of the glycans. The E proteins and pr domains are shown in green and orange respectively in all panels.
Supplementary Figure 4 Alignment of sequences that form the interfaces between immature ZIKV spikes.
Top panel, Alignment of pr domain sequences from different flaviviruses. The residues that differ between the pre-epidemic (African) and epidemic (Asian) ZIKV strains are indicated in blue boxes. Bottom panel, Alignment of parts of the E protein sequence from different flaviviruses. In both panels, red columns indicate strictly conserved residues, yellow columns show partially conserved residues and white columns show variable residues. Residues involved in interactions within a trimeric spike and between adjacent spikes are indicated by brown and green colored bars above the corresponding residues, respectively. The furin cleavage site at the end of the pr domain is shown by a grey bar. Abbreviations DEN, JEV and YFV refer to dengue virus, Japanese encephalitis virus and yellow fever virus respectively.
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Prasad, V., Miller, A., Klose, T. et al. Structure of the immature Zika virus at 9 Å resolution. Nat Struct Mol Biol 24, 184–186 (2017). https://doi.org/10.1038/nsmb.3352
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DOI: https://doi.org/10.1038/nsmb.3352
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