Thibaut Hendrik Jan, van der Linden Lonneke, Jiang Ping, Thys Bert, Canela María-Dolores, Aguado Leire, Rombaut Bart, Wimmer Eckard, Paul Aniko, Pérez-Pérez María-Jesús, van Kuppeveld Frank J M, Neyts Johan
Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium; Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium; Department Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
PLoS Pathog. 2014 Apr 10;10(4):e1004039. doi: 10.1371/journal.ppat.1004039. eCollection 2014 Apr.
Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.
肠道病毒（属于小核糖核酸病毒科）涵盖了一大类对医学具有重要意义的人类病原体，目前尚无获批的抗病毒治疗方法。尽管对这些病毒已进行了广泛研究，但病毒生命周期的某些方面，特别是形态发生，仍了解甚少。我们报告发现TP219是几种肠道病毒复制的新型抑制剂，包括柯萨奇病毒和脊髓灰质炎病毒。我们表明，TP219直接结合谷胱甘肽（GSH），从而迅速消耗细胞内GSH水平，并且这会干扰病毒形态发生而不影响病毒RNA复制。对组装的抑制作用表明不依赖于改变的还原环境。使用TP219，我们表明GSH是原聚体颗粒转变为五聚体颗粒过程中必不可少的稳定辅助因子。在低GSH水平存在的情况下，柯萨奇病毒B3的连续传代筛选出了GSH非依赖性突变体，这些突变体在两个原聚体之间的界面处的VP1中含有一个表面暴露的甲硫氨酸。与这一观察结果一致，已经含有这种表面暴露甲硫氨酸的肠道病毒，如EV71，在病毒形态发生过程中不依赖于GSH。生化和显微镜分析为GSH与野生型VP1之间的直接相互作用以及这种相互作用在将组装中间体定位到复制位点中的作用提供了有力证据。一致地，GSH与突变体VP1之间的相互作用被消除，导致组装中间体重新定位到独立于GSH的复制位点。因此，这项研究揭示了GSH是产生有感染性的肠道病毒子代所必需的新型稳定宿主因子，并为了解甚少的形态发生过程提供了新的见解。
