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.

IMPORTANCE Cucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus. 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.

黄瓜坏死病毒（CNV）是Tombusvirus属的成员，并且具有单部分正向RNA基因组。CNV在自然界中通过产生真菌Olpidiumbornovanus的游孢子传播。与汤姆巴斯病毒属的其他成员一样，CNV衣壳在暴露于碱性pH和EDTA时会溶胀。我们先前证明P73G突变可阻止病毒通过游动孢子传播，而不会显着影响植物的复制（K. Kakani，R。Reade和D. Rochon，J Mol Biol 338：507-517，2004，https：// doi .org / 10.1016 / j.jmb.2004.03.008）。P73紧邻假定的锌结合位点（M. Li等，J Virol 87：12166-12175，2013，https ://doi.org/10.1128/JVI.01965-13）是由三个二十面体相关的His残基在准6倍轴的C亚基的N末端形成的。为了更好地了解这种埋藏的残基如何影响载体的传递，我们确定了在自然条件下处于天然和溶胀状态的野生型CNV以及传递缺陷的突变体P73G的低温电子显微镜结构。对于野生型CNV，肿胀的结构表明衣壳的预期扩展。然而，在β-环轴线的准-6倍处的锌结合区保持完整。相比之下，即使在天然条件下，P73G突变体的锌结合区也明显紊乱，表明β环已被破坏，这可能使衣壳失去稳定性。pH和尿素变性实验结合电子显微镜分析证实了这一点。我们建议，P73G突变会影响锌的结合和/或β环带，使其在中性/碱性pH条件下更易碎。反过来，这可能会影响游动孢子的传播。

重要信息 黄瓜坏死病毒（CNV）是Tombusvirus属的一种，它是通过天竺葵（Ol​​pidiumbornovanus）真菌的游动孢子自然传播的。尽管许多植物病毒是通过昆虫载体传播的，但在分子水平上对于如何识别和传播病毒知之甚少。与许多球形植物病毒一样，当暴露于碱性pH和EDTA时，CNV衣壳会膨胀。我们以前证明了P73G突变位于衣壳内，紧邻假定的锌结合位点（Li等人，《病毒学杂志》（J Virol）87：12166-12175，2013，https：//doi.org/10.1128/JVI.01965- 13）阻止病毒通过游动孢子传播，而不会显着影响植物的复制（K.Kakani，R.Reade和D.Rochon，J Mol Biol 338：507-517，2004，https：//doi.org/10.1016/j .jmb.2004.03.008）。在这里，我们显示P73G突变体不如野生型稳定，这似乎与二十面体3倍轴处β环的不稳定相关。因此，β环空似乎不是粒子组装所必需的，而是与透射矢量相互作用所必需的。