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. 2016 Oct 4:7:13014.
doi: 10.1038/ncomms13014.

Structural insights into the assembly and regulation of distinct viral capsid complexes

Subir Sarker  1   2 , María C Terrón  3 , Yogesh Khandokar  4 , David Aragão  5 , Joshua M Hardy  6 , Mazdak Radjainia  6 , Manuel Jiménez-Zaragoza  7 , Pedro J de Pablo  7   8 , Fasséli Coulibaly  6 , Daniel Luque  3 , Shane R Raidal  1   2 , Jade K Forwood  2   4
Affiliations

Structural insights into the assembly and regulation of distinct viral capsid complexes

Subir Sarker et al. Nat Commun. .

Abstract

The assembly and regulation of viral capsid proteins into highly ordered macromolecular complexes is essential for viral replication. Here, we utilize crystal structures of the capsid protein from the smallest and simplest known viruses capable of autonomously replicating in animal cells, circoviruses, to establish structural and mechanistic insights into capsid morphogenesis and regulation. The beak and feather disease virus, like many circoviruses, encode only two genes: a capsid protein and a replication initiation protein. The capsid protein forms distinct macromolecular assemblies during replication and here we elucidate these structures at high resolution, showing that these complexes reverse the exposure of the N-terminal arginine rich domain responsible for DNA binding and nuclear localization. We show that assembly of these complexes is regulated by single-stranded DNA (ssDNA), and provide a structural basis of capsid assembly around single-stranded DNA, highlighting novel binding interfaces distinct from the highly positively charged N-terminal ARM domain.

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Figures

Figure 1
Figure 1. Structural characterization of two distinct BFDV-Cap complexes.
Left panel, negatively stained electron micrograph of the BFDV Cap protein shows two populations corresponding to VLPs (red box), and a smaller assembly of ∼10 nm in diameter (blue box). Right panel, X-ray crystal structures allow modelling of the two complexes to 2.0 Å (10 nm, top), and 2.5 Å (17 nm, bottom). The smaller complex is comprised of 10 Cap molecules arranged as two interlocking discs, with each disc containing five Cap molecules. The larger VLP is comprised of 12 pentamers arranged with T=1 icosahedral symmetry.
Figure 2
Figure 2. The N-terminal ARM domain accessibility is inverted in Cap complexes.
X-ray crystal structures reveal that the N-termini of all Cap molecules are positioned on the exterior and interior of the 10- and 60-mer complexes respectively (top panel). The accessibility of the highly positively charged N-terminal ARM domains containing both NLS and DNA-binding activity, modelled using I-tasser in blue cartoon, is confirmed in electron microscopy immunogold-labelling experiments of the N-terminal domain.
Figure 3
Figure 3. The population of the two viral complexes is regulated by ssDNA.
Negatively stained images of Cap complexes (see electron micrograph inserts in top panel) reveal 10-mer complexes are highly favoured in the absence of ssDNA, while ssDNA promotes 60-mer particle assembly. This is highly intuitive for the localization of Cap to the nucleus, as well as its interaction and packaging of the ssDNA viral genome. Synthesis of Cap molecules, occurring in the cytoplasm which is segregated from its ssDNA genome, require an accessible NLS and DNA-binding domains for interaction with nuclear import receptors and viral ssDNA respectively. Under these conditions, the 10-mer complexes are favoured over the 60-mer, which if formed in the cytoplasm would render these functional domains inaccessible, inhibiting nuclear localization and interaction with ssDNA. This is supported by Tobacco Etch Virus (TEV) and stability Assays (bottom panels), showing that in the absence of ssDNA (lane 1), the N-termini are exposed and susceptible to TEV proteolysis (left panel), and that the protein is less stable (right panel). Premature assembly of empty VLPs are limited in the absence of ssDNA due to the strong repulsive forces of the positively charged ARM domains. In the presence of ssDNA (lane 2), but not double-stranded DNA (lane 3) or plasmid DNA (lane 4), 60-mer particles are strongly favoured (see micrograph inserted in top panel). In the nucleus, the presence of ssDNA promotes the formation of 60-mer particles that are more stable, with the negatively charged ssDNA neutralizing the charge of the positively charged N-terminal ARM domains.
Figure 4
Figure 4. X-ray crystal and cryoEM structures of the Cap:ssDNA complex reveal novel ssDNA binding sites.
Top panel: Crystal diffraction of the Cap:ssDNA complex to 2.3 Å enabled modelling of 180 nucleotides (spheres) on the interior of the capsid (cartoon). Detailed interactions are presented in the top right panel, involving a range of side-chain interactions (presented as sticks). Residues listed with * and ** are from adjacent Cap monomers within and external to a pentameric protomer, respectively. A cryoEM reconstruction of the Cap:ssDNA complex (bottom panel) to 4.5 Å revealed density supporting the modelled ssDNA in the crystal structure, contiguous to the centre of the VLP (bottom panel). Electron density corresponding to the capsid protein is coloured in brown. The surface of the capsid interacting with the DNA is coloured in magenta. The remaining electron density is represented as a blue mesh both in the difference map (bottom left panel) and the full reconstruction (middle panel).
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