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. 2020 Feb 14;11(1):895.
doi: 10.1038/s41467-020-14647-9.

Capsid Protein Structure in Zika Virus Reveals the Flavivirus Assembly Process

Free PMC article

Capsid Protein Structure in Zika Virus Reveals the Flavivirus Assembly Process

Ter Yong Tan et al. Nat Commun. .
Free PMC article


Structures of flavivirus (dengue virus and Zika virus) particles are known to near-atomic resolution and show detailed structure and arrangement of their surface proteins (E and prM in immature virus or M in mature virus). By contrast, the arrangement of the capsid proteins:RNA complex, which forms the core of the particle, is poorly understood, likely due to inherent dynamics. Here, we stabilize immature Zika virus via an antibody that binds across the E and prM proteins, resulting in a subnanometer resolution structure of capsid proteins within the virus particle. Fitting of the capsid protein into densities shows the presence of a helix previously thought to be removed via proteolysis. This structure illuminates capsid protein quaternary organization, including its orientation relative to the lipid membrane and the genomic RNA, and its interactions with the transmembrane regions of the surface proteins. Results show the capsid protein plays a central role in the flavivirus assembly process.

Conflict of interest statement

The authors declare no competing interests.


Fig. 1
Fig. 1. Capsid protein topology and sequence.
a Topology of the translated single ZIKV polyprotein chain. The full-length capsid protein, for which the cryoEM structure is determined here, is highlighted in dark gray. b Sequence alignment of capsid proteins across flaviviruses. Above the amino acid sequences, the green rectangles represent the helical structures, whereas the gray lines represent the loops in the capsid structure of our cryoEM structure of immZIKV complexed with DV62.5. Dotted lines indicate parts that could not be observed. Similar coloring codes were used to box the sequences of residues forming the secondary structures observed in the crystal structures of the capsid proteins of ZIKV (PDB ID:5YGH) and WNV (PDB ID: 1SFK), and NMR structure of the capsid protein of DENV (PDB ID: 1R6R). The NS2B/NS3 and the signal peptidase cleavage sites, are also indicated. Two proline residues that interrupt the formation of helix α1 leading to a shorter version in the ZIKV compared with the other flavivirus are indicated by red asterisks.
Fig. 2
Fig. 2. ImmZIKV structure is stabilized by DV62.5, improving resolution of the capsid protein density.
The surface (top panels) and a quarter of a central slice (bottom panels) of the cryoEM maps of a immZIKV:DV62.5 and b uncomplexed immZIKV. The central slice shows the capsid protein bridges the RNA and the inner leaflet of the lipid membrane. The overall resolution of the maps (top) and the capsid layer regions (bottom) are shown. Densities corresponding to Fabs are colored in orange. c Overall structure of a capsid dimer with each protomer containing helices α1 to α5 (colored from light to dark brown shades). Stereo-diagram (two left panels) of the fitted capsid dimer (ribbon) in its corresponding density (gray) at a contour level of 1.65. Different views of capsid dimer structure (two right panels) are shown. The N terminus (red N letter) and C terminus (blue C letter) of the capsid protein are indicated. One of protomer is colored in gray. d Occupancies of the Fab DV62.5 on the immZIKV. Left panel, on immZIKV, Fab DV62.5 (gray surface representation) binds to all red prM–E complex. The epitope on the blue prM–E is completely occluded by neighboring prM–E molecules; therefore, no Fab binding is detected. Around the threefold axis, although the epitope is completely exposed in all green prM–E-protein molecules, the space at threefold vertices is likely unable to accommodate three Fabs. E–prM proteins with bound Fab DV62.5 at full occupancy are indicated by asterisks (*), whereas those with no binding by crosses (×). The three individual E proteins in an asu, each located adjacent to either five-, two- or threefold vertices, are colored in red, green, and blue, respectively, and their binding partner, the prM molecule in a lighter shade of the same color. e Localized reconstruction on the densities surrounding the threefold vertices showed two major Fab binding classes. Left panel, the first class of Fab binding shows only one Fab (yellow) around this vertex. Right panel, the second class of Fab binding showed two Fabs bound (yellow and magenta). The E–prM proteins and Fab molecules are drawn in ribbon representation.
Fig. 3
Fig. 3. The helices α5 are important for facilitating trimerization of capsid dimers.
a The fit of crystal structure of ZIKV capsid protein dimers (dotted black circles) into the immZIKV density map (transparent gray). b Two capsid dimers interact via their hydrophobic interactions between helices α5. c Side view showing the orientation of the capsid protein with respect to the lipid bilayer membrane and the viral RNA. The capsid dimer is located below the cluster of the prM and E-TM regions. One capsid protein contains five helices (α1–α5). The helices of one capsid protomer within the dimer are colored from the lightest to the darkest shade of brown, whereas the other capsid protomer is colored in light gray. The helix α1 of both protomers clustered together forming a largely hydrophobic surface interacting with the viral lipid membrane. The helix α4 containing highly positively charged residues facing the negatively charged RNA. d View from the inside of the virus, three capsid protein dimers interact with each other via helix α5 forming a triangular network.
Fig. 4
Fig. 4. Presence of helix α5 and its importance on capsid protein dimers oligomerization.
a The higher MW capsid protein contains helix α5. The viral protease NS2B/NS3 is known to cleave full-length capsid protein between helix α4 and α5. Tartrate-purified ImmZIKV was lysed with 1% DDM and incubated with native or heat-inactivated NS2B/NS3 followed by western blotting analysis with anti-ZIKV capsid antibody. In the absence of or with heat-inactivated NS2B/NS3, two capsid bands were detected, suggesting two population of capsid proteins—likely capsid with and without helix α5. In the presence of NS2B/NS3, the intensity of the higher molecular weight capsid band decreased, whereas that of the lower band increased, suggesting that the upper band contains helix α5. b Helix α5 is essential for the trimerization of capsid protein dimers. SDS-PAGE western blot analysis of boiled ( + ) or not boiled ( − ) samples containing purified MBP only, MBP fused to capsid protein with and without helix α5, using an anti-MBP antibody. MBP:capsid protein with helix α5 shows the presence of dimer of dimers and trimer of dimers, and when helix α5 is absent the capsid protein is largely unable to form these higher oligomerization states. c Native PAGE analysis of MBP-fused capsid proteins showed that the full-length capsid proteins with helix α5 can form a higher oligomeric band but not the capsid proteins without helix α5. d Capsid helix α5 may not be stable in the ER membrane. Comparison of the TM region of the E protein (left) with the capsid helix α5 (right). The dotted line indicates the boundary of the hydrophobic region. This shows that the helix α5 is much shorter than that of the E-TM region, suggesting it is likely to be less stable in the ER membrane and thus could be pulled out of the membrane when the capsid protein interacts with RNA. Signal peptidase cleavage site is indicated.
Fig. 5
Fig. 5. Capsid protein is important for the organization of the virus quaternary structure.
a Capsid protein dimer is located in between the fivefold and threefold vertices (left and middle top panels); it acts as a base supporting the red, green, and blue prM–E-protein complexes (bottom panel). Right-top panel, one capsid dimer likely interacts with the loop that links the two anti-parallel helices of the TM regions of three prM proteins and the green E protein (pink dotted circles). b View from the inside of the virus, three capsid protein dimers form a triangular network around the threefold vertices. Neighboring capsid dimer triangular networks around other threefold vertices are colored in black and the prM–E complexes as gray surfaces. The dotted orange box indicates the same location as in a, but viewed from the inside of the virus. c Each capsid protein dimer binds to three E–prM protein complexes (circled in dotted or dash, or black lines); therefore, one triangular network of capsid protein dimers brings together a total of nine E–prM heterodimers, forming an assembly unit.
Fig. 6
Fig. 6. Proposed assembly process of immZIKV particle.
a Capsid proteins are expressed in the cytoplasmic side of the ER membrane, where they form homodimers. One side of the homodimer interacts with ER membrane, whereas the other side with viral RNA. Simultaneously, on the luminal side of the ER, the prM and E proteins form heterodimers and then three of these heterodimers associate with each other to form an inverted tripod. b The inverted tripods interact with each other. The capsid dimers then attach to the TM regions of the prM/E tripods. c The capsid dimers then interact with other nearby capsid dimers via their helix α5. This sets the spacing between the inverted tripods. Three prM/E inverted tripods with capsid protein dimers binding underneath form an assembly unit. d The tips of these assembly units (pink boxes) then interact to form a virus surface lattice on the lumen side of the ER membrane. One assembly unit is colored in gray and the other in yellow.

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