To Janet, Surya Wahyu, Fung To Sing, Li Yan, Verdià-Bàguena Carmina, Queralt-Martin Maria, Aguilella Vicente M, Liu Ding Xiang, Torres Jaume
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
Department of Physics, Laboratory of Molecular Biophysics. Universitat Jaume I, Castelló, Spain.
J Virol. 2017 Feb 14;91(5). doi: 10.1128/JVI.02158-16. Print 2017 Mar 1.
It has been shown previously in the severe acute respiratory syndrome coronavirus (SARS-CoV) that two point mutations, N15A and V25F, in the transmembrane domain (TMD) of the envelope (E) protein abolished channel activity and led to attenuation. Pathogenicity was recovered in mutants that also regained E protein channel activity. In particular, V25F was rapidly compensated by changes at multiple V25F-facing TMD residues located on a neighboring monomer, consistent with a recovery of oligomerization. Here, we show using infected cells that the same mutations, T16A and A26F, in the gamma-CoV infectious bronchitis virus (IBV) lead to, in principle, similar results. However, IBV E A26F did not abolish oligomer formation and was compensated by mutations at N- and C-terminal extramembrane domains (EMDs). The C-terminal EMD mutations clustered along an insertion sequence specific to gamma-CoVs. Nuclear magnetic resonance data are consistent with the presence of only one TMD in IBV E, suggesting that recovery of channel activity and fitness in these IBV E revertant mutants is through an allosteric interaction between EMDs and TMD. The present results are important for the development of IBV live attenuated vaccines when channel-inactivating mutations are introduced in the E protein. The ion channel activity of SARS-CoV E protein is a determinant of virulence, and abolishment of channel activity leads to viral attenuation. E deletion may be a strategy for generating live attenuated vaccines but can trigger undesirable compensatory mechanisms through modifications of other viral proteins to regain virulence. Therefore, a more suitable approach may be to introduce small but critical attenuating mutations. For this, the stability of attenuating mutations should be examined to understand the mechanisms of reversion. Here, we show that channel-inactivating mutations of the avian infectious bronchitis virus E protein introduced in a recombinant virus system are deficient in viral release and fitness and that revertant mutations also restored channel activity. Unexpectedly, most of the revertant mutations appeared at extramembrane domains, particularly along an insertion specific for gammacoronaviruses. Our structural data propose a single transmembrane domain in IBV E, suggesting an allosteric interaction between extramembrane and transmembrane domains.
先前已在严重急性呼吸综合征冠状病毒（SARS-CoV）中表明，包膜（E）蛋白跨膜结构域（TMD）中的两个点突变N15A和V25F消除了通道活性并导致病毒减毒。在也恢复了E蛋白通道活性的突变体中恢复了致病性。特别是，V25F被位于相邻单体上的多个面向V25F的TMD残基的变化迅速补偿，这与寡聚化的恢复一致。在这里，我们使用感染细胞表明，γ冠状病毒传染性支气管炎病毒（IBV）中的相同突变T16A和A26F原则上导致类似结果。然而，IBV E A26F并没有消除寡聚体形成，而是通过N端和C端膜外结构域（EMD）的突变得到补偿。C端EMD突变沿着γ冠状病毒特有的插入序列聚集。核磁共振数据与IBV E中仅存在一个TMD一致，这表明这些IBV E回复突变体中通道活性和适应性的恢复是通过EMD和TMD之间的变构相互作用实现的。当在E蛋白中引入通道失活突变时，本研究结果对于IBV减毒活疫苗的开发具有重要意义。SARS-CoV E蛋白的离子通道活性是毒力的决定因素，通道活性的消除导致病毒减毒。E基因缺失可能是产生减毒活疫苗的一种策略，但可能会通过其他病毒蛋白的修饰引发不良的补偿机制以恢复毒力。因此，一种更合适的方法可能是引入小的但关键的减毒突变。为此，应检查减毒突变的稳定性以了解回复机制。在这里，我们表明，在重组病毒系统中引入的禽传染性支气管炎病毒E蛋白的通道失活突变在病毒释放和适应性方面存在缺陷，并且回复突变也恢复了通道活性。出乎意料的是，大多数回复突变出现在膜外结构域，特别是沿着γ冠状病毒特有的插入序列。我们的结构数据表明IBV E中存在单个跨膜结构域，这表明膜外结构域和跨膜结构域之间存在变构相互作用。
