|Entry||Database: EMDB / ID: 8818|
|Title||Cryo-EM structure of the P73G mutant of cucumber necrosis virus under native conditions|
|Map data||Cucumber necrosis virus, P73G mutant. Mutation blocks insect vector transmission but is buried in the capsid. Map shows disruption in the putative Zn binding site that may explain loss of transmission.|
|Sample||Cucumber necrosis virus:|
|Function / homology||Icosahedral viral capsid protein, S domain / Viral coat protein subunit / Viral coat protein (S domain) / Plant viruses icosahedral capsid proteins 'S' region signature. / T=3 icosahedral viral capsid / structural molecule activity / RNA binding / Capsid protein|
Function and homology information
|Source||Cucumber necrosis virus|
|Method||single particle reconstruction / cryo EM / 4.2 Å resolution|
|Citation||Journal: J. Virol. / Year: 2017|
Title: Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission.
Authors: Michael B Sherman / Kishore Kakani / D'Ann Rochon / Wen Jiang / Neil R Voss / Thomas J Smith
Abstract: (CNV) is a member of the genus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus As with other members of the genus, the CNV capsid swells ... (CNV) is a member of the genus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus As with other members of the genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the β-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the β-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the β-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission. (CNV), a member of the genus , is transmitted in nature via zoospores of the fungus While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166-12175, 2013, https://doi.org/10.1128/JVI.01965-13) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507-517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the β-annulus at the icosahedral 3-fold axes. Therefore, the β-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.
|Date||Deposition: Jul 10, 2017 / Header (metadata) release: Jul 19, 2017 / Map release: Jul 19, 2017 / Last update: Sep 27, 2017|
|Structure viewer||EM map: |
Downloads & links
|File||emd_8818.map.gz (map file in CCP4 format, 665501 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.03 Å|
CCP4 map header:
-Entire Cucumber necrosis virus
|Entire||Name: Cucumber necrosis virus / Number of components: 1|
-Component #1: virus, Cucumber necrosis virus
|Virus||Name: Cucumber necrosis virus / Class: VIRION / Empty: No / Enveloped: No / Isolate: STRAIN|
|Species||Species: Cucumber necrosis virus|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||pH: 5|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
|Imaging||Microscope: JEOL 2200FS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 37 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Camera||Detector: DIRECT ELECTRON DE-20 (5k x 3k)|
|Processing||Method: single particle reconstruction / Number of projections: 49525|
|3D reconstruction||Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF|
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