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Thomas Smith's Laboratory |
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The N-terminus of CNV controls capsid assembly
Despite having only 19% capsid protein sequence identity (34% similarity) to
cowpea chlorotic mottle virus (CCMV), the core structures of these two members
of the Bromoviridae family are highly homologous. In CCMV, the structures of the A, B, and C subunits are nearly
identical except for the N-terminus. In contrast, the structures of two loops in
subunit A of CMV differ from those in B and C. These loops are 6 and 7 residues
longer than the analogous regions in CCMV. Unlike CCMV, the capsid of CMV does
not undergo swelling at pH 7.0 and is stable at pH 9.0. This may be partly due
to the fact that the N-termini of the B and C subunits form a unique bundle of 6
amphipathic helices orientated down into the virion core at the 3-fold axes. In
addition, while CCMV has a cluster of aspartic acid residues at the quasi
three-fold axis that are proposed to bind metal in a pH dependent manner, this
cluster is replaced by complementing acids and bases in CMV. Finally, this
structure clearly demonstrates that the residues important for aphid
transmission lie at the outer-most portion of the ßH-ßI loop and yields details
of the portions of the virus that are hypothesized to mediate binding to the
aphid mouthparts.
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CMV organization
On the left is a schematic representation of the T=3,
truncated icosahedron. The A,B,C denote the three identical subunits and the
black dots represent the N-terminal alpha helices. On the right is a surface representation of the CMV caspid colored
according to radial distance.
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Identification of a loop crucial for aphid transmission.
On the left is a C-alpha backbone of one of the
three subunits. The coloring scheme is where the color ramps from red to blue as
it extends from the N to C termini. The residues denoted by the mauve balls are
those which, when mutated, affect the ability of aphids to transmit the viruses
as shown in the table on the right. As shown below, this same loop appears to be
binding a metal ion. On the right are the effects of the various mutations in this loop on aphid transmission.
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Virus |
Aphid Transmission
(# infected/total) |
Wt |
70/70 |
D191A |
12/24 |
D191K |
0/17 |
D192A |
0/30 |
D192K |
8/27 |
L194A |
19/28 |
E195A |
3/22 |
E195K |
11/45 |
D197A |
12/45 |
D197K |
20/20 |
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Structure of an antibody/CMV complex
In order to further study the aphid transmission site, we determined the structure of an Fab/CMV complex where the antibody was able to bind to the native virus but unable to bind to the aphid transmission mutants. A) The image reconstruction of the complex. Arrow 3 is the location of the variable domains and 4 the constant domains. Note that antibodies only bind to pentons, yet all subunits are chemicall identical. Arrow 1 in (D) shows the unusual channel formed by the helical bundle at the quasi 6-fold axes. Figure (B) shows the surface of the model were the Fab is placed at all possible positions at the 5-fold (each with a 1/5 occupancy) since there is only room for 1 Fab to bind at a time.
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Fab contacts at the 5-fold axes
Shown here are the contacts made by one Fab binding at the pento (A). Note how each Fab binds several subunits at a time - a true 3D epitope. B) This figure shows the location of the aphid transmission loop described above. Note that the Fab does indeed contact this loop. C) This figure shows why an antibody binding can bind to the 5-fold but not the quasi 6-fold axes. The white area shows the contact region shown in (A) but how the distribution of these contacts are totally different at the quasi 6-fold axes. Therefore, even though the subunits at these axes are the same in structure and chemical identity, their quaternary assembly is very different and the antibody is seeing these differences. |
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Our relevant publications:
- Bowman, V. D., Chase, E. S., Franz, A. W. E., Chipman, P. R., Zhang, X., Perry, K. L., Baker, T. S., Smith, T. S. (2002) An antibody to the putative aphid recognition site on cucumber mosaic virus recognizes pentons but not hexons. J. Virol 76:12250-12258.
- Smith, T. J., Chase, E., Schmidt, T., Perry, K (2000) The structure of cucumber mosaic virus and comparison to cowpea chlorotic mottle virus. J. Virol. 74: 7578-7586.
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