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Thomas Smith's Laboratory |
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The N-terminus of CNV controls capsid assembly
Shown here is the C subunit of TBSVand CNV R domain regions. The structure of the C subunit of TBSV with the arm, S, and P domains colored red, blue, and green, respectively, is shown at the top. The entire arm domain is disordered in the A and B subunits, whereas it forms the β-annulus at the 3-fold axes in the C subunit. Also noted here is the location of the proline residue (P85 in CNV) that, upon mutation to glycine in some of the CNV constructs, changes the assembly equilibrium to favor the formation of T=1 capsids. The 58 amino acid sequence of the 58 CNV R domain, and the three subdomains that influence capsid formation are shown at the bottom. The sequence at the bottom is not observed in the crystal structure and is often thought to be interacting with the RNA interior. While it is often thought that these interactions are dominated by basic residues in the protein, it is clear from character of regions 1 and 2 that this is not entirely the case. By altering these three regions alone or in concert affects the assembly of the large T3 particles and small, T1 particles are produced.
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Image reconstruction of cucumber necrosis virus
We determined the structure of the T1 and T3 CNV particles to 11Å using cryo-EM methods. As shown below, the CNV structure (left) closely matches that of another tombusvirus, TBSV (right). In the figure on the right, the A, B, and C subunits are colored blue, green, and red, respectively.
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Possible internal structures in the CNV T3 particles.
When the internal aspects of the image reconstruction was examined, several unusual features were observed. The outer shell (right, blue) closely matched that of TBSV. Immediately beneath that was a rather amorphous shell of density (right, mauve). Finally, inside of that shell was a very unusual feature that looked like a 'mini virus' with flat faces and it appeared to have connections with the outer shell (right, orange). The dimensions of these shells closely matched previous neutron diffraction results by other groups (left figure) that suggested that the mauve shell is RNA and the inner orange shell is protein. Perhaps the N-terminal region, necessary for T3 capsid formation, forms a sort of internal scaffold for virus assembly. |
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Image reconstuction of the CNV T1 particle
As mentioned above, mutation of the N-terminal regions of CNV results in small T1 particles rather than the larger T3 particles (60 copes of capsid protein rather than 180). As shown below, the image reconstruction (left image) is well described by a model of TBSV where the B and C subunits are removed and the pentons are moved to a smaller radius (right image). It is important to note that none of the unusual internal structure seen in the T3 particles are not observed here. |
Stereo images of CNV
Shown here is a red/blue stereo image of CNV (left) and a cutaway view of the T3 particle (right). In the cutway view, the preported RNA shell has been removed to better show the unusual internal feature that neutron scattering suggests is protein. |
Our relevant publications:
- Kakani, K., Reade, R., Katpally, U., Smith, T. J., Rochon, D. (2008) Induction of particle polymorphism by Cucumber necrosis virus coat protein in vivo. J. Virol. 82:1457-1557.
- Katpally, U., Kakani, K., Reade, R., Rochon, D., Smith, T. J. (2007) Structures of T=1 and T=3 Cucumber necrosis particles: evidence of internal scaffolding. J. Mol. Biol. 365:502-512.
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