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Chikungunya virus assembly and budding visualized in situ using cryogenic electron tomography

Abstract

Chikungunya virus (CHIKV) is a representative alphavirus causing debilitating arthritogenic disease in humans. Alphavirus particles assemble into two icosahedral layers: the glycoprotein spike shell embedded in a lipid envelope and the inner nucleocapsid (NC) core. In contrast to matrix-driven assembly of some enveloped viruses, the assembly/budding process of two-layered icosahedral particles remains poorly understood. Here we used cryogenic electron tomography (cryo-ET) to capture snapshots of the CHIKV assembly in infected human cells. Subvolume classification of the snapshots revealed 12 intermediates representing different stages of assembly at the plasma membrane. Further subtomogram average structures ranging from subnanometre to nanometre resolutions show that immature non-icosahedral NCs function as rough scaffolds to trigger icosahedral assembly of the spike lattice, which in turn progressively transforms the underlying NCs into icosahedral cores during budding. Further, analysis of CHIKV-infected cells treated with budding-inhibiting antibodies revealed wider spaces between spikes than in icosahedral spike lattice, suggesting that spacing spikes apart to prevent their lateral interactions prevents the plasma membrane from bending around the NC, thus blocking virus budding. These findings provide the molecular mechanisms for alphavirus assembly and antibody-mediated budding inhibition that provide valuable insights for the development of broad therapeutics targeting the assembly of icosahedral enveloped viruses.

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Fig. 1: CHIKV assembly and budding at the infected-cell periphery.
Fig. 2: Classification and enumeration of CHIKV budding intermediates.
Fig. 3: In situ refined structures of CHIKV assembly intermediates after extensive subtomogram classification and averaging.
Fig. 4: CHIKV envelope spikes arranged in hexagonal lattices form helical tubes in situ.
Fig. 5: Neutralizing antibody C9 crosslinks spikes at the cell surface and induces coalescence of spike-C9 complexes.
Fig. 6: Mechanistic model of alphavirus budding and assembly.

Data availability

Cryo-EM maps reported in this study have been deposited in the Electron Microscopy Data Bank (EMDB) under the following accession codes: EMDB-26446 (released virion), EMDB-26447, −26448, −26449, −26450 (budding intermediates) and EMDB-26451, −26452 (cytosolic NLPs). The publicly deposited atomic model of VEEV TC-83 (PDB:3J0C) was used for comparison to the subtomogram average structure of the CHIKV spike trimer determined in this study. All other data supporting the findings of this study are available within the Article and its supplementary files.

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Acknowledgements

We thank SLAC National Accelerator Laboratory for access and support of these studies, and all SLAC cryo-EM staff for technical support and assistance. We also thank M. Chen for helpful discussions and providing technical advice on data analysis. This research was supported by NIH grants R01AI148382 and S10OD021600 (to W.C.) and R01AI119056 (to G.S.).

Author information

Authors and Affiliations

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Contributions

D.C., J.J. and W.C. designed the study. D.C and J.J. performed cryo-EM sample preparation and collected cryo-ET data. D.C. performed 3D reconstruction and subtomogram averaging. D.C., J.J., M.F.S. and W.C. analysed the data. D.C., J.J. and W.C. wrote the manuscript with support from all co-authors.

Corresponding authors

Correspondence to Jing Jin or Wah Chiu.

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The authors declare no competing interests.

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Nature Microbiology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 RNA replication spherules on the cell surface.

(a) Tomogram slice displaying cell periphery with RNA replication spherules (yellow arrows), budding intermediates (white arrows) and apparently cytosolic NLPs (red arrows). Scale bar: 100 nm. (b) Enlarged tomogram slice of RNA replication spherule with components labeled. Proposed location of the replication complex at the neck of spherules was indicated with a yellow dashed circle. (c) Enlarged slice view of a RNA spherule with a NLP (red dashed circle) in close proximity to the spherule neck (yellow dashed circle), with some thin density (blue arrow) connecting in between. (d) Enlarged slice view of RNA spherule in close proximity to another apparently incompletely-assembled NLP (red dashed circle) and multiple budding particles (white arrows).

Extended Data Fig. 2 Strands of incomplete particles extend from the cell surface.

(a-d) Tomogram slice images display thin extensions of virus budding intermediates in multiple conformations (white dashed boxes, inset images). In all observed cases, beads of linked particles form at the convergences of two membrane surfaces with near-continuous budding intermediates. Scale bars: 200 nm.

Extended Data Fig. 3 CryoET data processing workflow and resolutions of STA structures.

(a) CryoET and data processing workflow for release virions (left), budding intermediates (middle) and budding arrested NLPs (right). For budding intermediates subvolume classification (middle), particles in 5 of the 10 classes (dashed black boxes) from the first multi-reference refinement were subjected to additional classification. 12 distinct intermediate budding conformations (blue boxes) were resolved after two rounds of classification. Note: two maps displayed within a single blue box merged into one class due to overall structure similarity. (b) Gold standard Fourier shell correlation (FSC) plots of subtomogram average structures of budding intermediates and released virions. (c) Slice view of 3D reconstruction of released CHIKV virions. Subnanometer (8.3 Å) resolution of structure is evident by resolved TM helices of E1/E2 in the lipid bilayer. (d) Central section of released icosahedral CHIKV particle reveals density of CP C-protease domain (red) as well as an ordered network of density layer below (purple) that is likely CP N-terminus+gRNA.

Extended Data Fig. 4 Non-icosahedral features of virus particles.

(a) Subtomogram average structures of cytosolic NLPs and NCs from budding-intermediate 3D reconstructions (shown in red). NC structures are overlaid on the density map of the NC from released CHIKV virion (white, transparent). (b) Volta phase plate tomogram slice images of PM with multiple budding intermediates, including latestage (‘tethered’) particles. Inset is the zoom-in view of the boxed area in (B) displaying relatively absent density at the base of latestage budding particles (blue arrow). Top of a budding particle, furthest from PM is defined as the leading end, while base of the particle is defined as the trailing end. (c) Subtomogram average structure of ‘tethered intermediate’ shows icosahedral symmetry at the leading end, while trailing end of average shows a disordered final penton and distorted capsomer density in those hexamer units below. Relatively absent density between NC and viral envelope (blue arrows) was also observed at (d) the non-spherical pole of released virions, and (e) the released multi-cored particles. NCs in multi-core particles are labeled with dashed red circles.

Extended Data Fig. 5 Orientation of cytosolic NLPs.

(a) Volta-phase plate tomogram slice image shows budding cell periphery with apparently cytosolic NLPs (red arrows). Scale bar: 50 nm. (b,c) Zoom-in views of NLPs (red arrows). Scale bars: 30 nm. (d) Tomogram slice image of cell with subvolume averages of NLP class II (red, Extended Data Fig. 4) mapped back to the tomogram based on the refined orientation of each particle. 5-fold density consistently oriented towards the PM surface in slices above. (e) Tomogram slice image displays cluster of NLPs (red arrow). Scale bar: 50 nm. (f) In a tomogram slice directly above a single NLP (white box), a penton of spikes (dashed white circle) is identified along with nearby spikes (white arrows). Scale bar: 50 nm.

Extended Data Fig. 6 Different assemblies of spikes on virus-infected cell surface.

(A) Single slice of tomogram depicting virus-induced extension with multiple spikeinduced features, including (I) hexameric sheet lattice, (II) budding strand of intermediates, and (III) helical tube. Scale bar 200 nm. (B.I) Tomographic slice view of near-planar lattice (A-I) of trimeric spikes arranged as hexamers (orange) and disrupted region (yellow). (B.II) 3D annotation of spikes (orange) shows disruption of hexameric lattice above NC (A-yellow arrow). (C.I) Tomographic slice view of budding intermediate particle surface (A-II) shows pentamer (yellow) and hexamer (orange) of trimeric spikes. (C.II) 3D annotation of particles on budding strand displays spherical curvature of spike lattice with pentamer (yellow arrow) and hexamer (black arrow). (D.I) Slice view of helical tube (A-III) formed by hexameric lattice of spikes with (D.II) 3D annotation.

Extended Data Fig. 7 Self-assembled structures of spikes alone.

(a-c) Images of CHIKV-infected cell peripheries display cell extensions (white arrows) directly from the cell body. Scale bars: 1 μm. (d & e) Zoom-in images of the boxed regions in B & C, display tubular protein arrays (black arrows) at the terminal end and released vesicle-like assembly products lacking dense cores (white arrows). Scale bars: 200 nm. (f & g) 3D segmentations of cellular features corresponding to (D & E) show the surfaces of tubular extensions contain dense spikes. Segmentations reveal spikes on the surface of tubular membrane extensions that largely exclude bundled cytoskeleton filaments.

Extended Data Fig. 8 Different conformations of spikes before and after assembly in budding shells.

(a,d) Tomogram slice displaying cell periphery with budding intermediates. Scale bars: 200 nm. (b, c, e, f) Enlarged tomogram slices of budding particles. Spike side-views on budding intermediates surface (blue dashed circle) displayed characteristic conformation of spikes assembled in lattice, in contrast to potential individual spikes (yellow arrows) near the base of budding intermediates.

Supplementary information

Supplementary Information

Supplementary Tables 1–2.

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Supplementary Video 1

A representative cryo-electron tomogram of a CHIKV-infected U2OS cell.

Supplementary Video 2

A representative cryo-electron tomogram of a NAb C9-treated CHIKV-infected U2OS cell

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Chmielewski, D., Schmid, M.F., Simmons, G. et al. Chikungunya virus assembly and budding visualized in situ using cryogenic electron tomography. Nat Microbiol 7, 1270–1279 (2022). https://doi.org/10.1038/s41564-022-01164-2

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