Stability of cucumber necrosis virus at the quasi-6-fold axis affects zoospore transmission

Michael Sherman, Kishore Kakani, D'Ann Rochon, Wen Jiang, Neil R. Voss, Thomas Smith

Research output: Contribution to journalArticle

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Abstract

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. As with other members of the Tombusvirus 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.

Original languageEnglish (US)
Article numbere01030-17
JournalJournal of Virology
Volume91
Issue number19
DOIs
StatePublished - Oct 1 2017

Fingerprint

Cucumber necrosis virus
Cucumis sativus
zoospores
Necrosis
capsid
Viruses
Tombusvirus
zinc
Zinc
Capsid
Olpidium
mutation
mutants
Cryoelectron Microscopy
Mutation
denaturation
binding sites
electron microscopy
Edetic Acid
urea

Keywords

  • Cryo-EM
  • Protein structure-function
  • RNA virus

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Stability of cucumber necrosis virus at the quasi-6-fold axis affects zoospore transmission. / Sherman, Michael; Kakani, Kishore; Rochon, D'Ann; Jiang, Wen; Voss, Neil R.; Smith, Thomas.

In: Journal of Virology, Vol. 91, No. 19, e01030-17, 01.10.2017.

Research output: Contribution to journalArticle

Sherman, Michael ; Kakani, Kishore ; Rochon, D'Ann ; Jiang, Wen ; Voss, Neil R. ; Smith, Thomas. / Stability of cucumber necrosis virus at the quasi-6-fold axis affects zoospore transmission. In: Journal of Virology. 2017 ; Vol. 91, No. 19.
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AB - Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. As with other members of the Tombusvirus 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.

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