Department of Biology, Indiana University, Bloomington, Indiana, USA.
Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, USA.
J Virol. 2022 Mar 9;96(5):e0214921. doi: 10.1128/jvi.02149-21. Epub 2022 Jan 12.
Alphaviruses are enveloped viruses transmitted by arthropod vectors to vertebrate hosts. The surface of the virion contains 80 glycoprotein spikes embedded in the membrane, and these spikes mediate attachment to the host cell and initiate viral fusion. Each spike consists of a trimer of E2-E1 heterodimers. These heterodimers interact at the following two interfaces: (i) the intradimer interactions between E2 and E1 of the same heterodimer and (ii) the interdimer interactions between E2 of one heterodimer and E1 of the adjacent heterodimer (E1'). We hypothesized that the interdimer interactions are essential for trimerization of the E2-E1 heterodimers into a functional spike. In this work, we made a mutant virus (chikungunya piggyback [CPB]) where we replaced six interdimeric residues in the E2 protein of Sindbis virus (wild-type [WT] SINV) with those from the E2 protein from chikungunya virus and studied its effect in both mammalian and mosquito cell lines. CPB produced fewer infectious particles in mammalian cells than in mosquito cells, relative to WT SINV. When CPB virus was purified from mammalian cells, particles showed reduced amounts of glycoproteins relative to the capsid protein and contained defects in particle morphology compared with virus derived from mosquito cells. Using cryo-electron microscopy (cryo-EM), we determined that the spikes of CPB had a different conformation than WT SINV. Last, we identified two revertants, E2-H333N and E1-S247L, that restored particle growth and assembly to different degrees. We conclude the interdimer interface is critical for spike trimerization and is a novel target for potential antiviral drug design. Alphaviruses, which can cause disease when spread to humans by mosquitoes, have been classified as emerging pathogens, with infections occurring worldwide. The spikes on the surface of the alphavirus particle are absolutely required for the virus to enter a new host cell and initiate an infection. Using a structure-guided approach, we made a mutant virus that alters spike assembly in mammalian cells but not mosquito cells. This finding is important because it identifies a region in the spike that could be a target for antiviral drug design.
甲病毒是通过节肢动物媒介传播给脊椎动物宿主的包膜病毒。病毒粒子的表面含有 80 个嵌入膜中的糖蛋白刺突，这些刺突介导与宿主细胞的附着并启动病毒融合。每个刺突由三聚体的 E2-E1 异二聚体组成。这些异二聚体在以下两个界面上相互作用：(i) 同一异二聚体的 E2 和 E1 之间的单体内相互作用，以及 (ii) 一个异二聚体的 E2 与相邻异二聚体 (E1') 的 E1 之间的单体间相互作用。我们假设单体间相互作用对于 E2-E1 异二聚体三聚化为功能性刺突是必不可少的。在这项工作中，我们构建了一种突变病毒（基孔肯雅猪驮病毒 [CPB]），其中我们用基孔肯雅病毒的 E2 蛋白中的六个单体间残基替换了辛德毕斯病毒（野生型 [WT] SINV）的 E2 蛋白中的相应残基，并在哺乳动物和蚊子细胞系中研究了其效应。与 WT SINV 相比，CPB 在哺乳动物细胞中产生的感染性颗粒比在蚊子细胞中少。当 CPB 病毒从哺乳动物细胞中纯化时，与源自蚊子细胞的病毒相比，颗粒中的糖蛋白含量相对减少，并且颗粒形态存在缺陷。使用冷冻电镜 (cryo-EM)，我们确定 CPB 的刺突具有与 WT SINV 不同的构象。最后，我们鉴定了两个回复突变体，E2-H333N 和 E1-S247L，它们在不同程度上恢复了颗粒的生长和组装。我们得出结论，单体间界面对于刺突三聚化至关重要，是潜在抗病毒药物设计的新靶点。当蚊子传播给人类时，甲病毒会引起疾病，因此被归类为新兴病原体，感染发生在世界各地。病毒粒子表面的刺突对于病毒进入新宿主细胞并引发感染是绝对必需的。我们使用结构导向方法构建了一种突变病毒，该病毒改变了哺乳动物细胞中的刺突组装，但不改变蚊子细胞中的刺突组装。这一发现很重要，因为它确定了刺突中可以作为抗病毒药物设计靶点的区域。
