Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, France.
Synchrotron SOLEIL, France.
PLoS Pathog. 2020 Mar 9;16(3):e1008383. doi: 10.1371/journal.ppat.1008383. eCollection 2020 Mar.
Mokola virus (MOKV) belongs to the lyssavirus genus. As other genus members-including rabies virus (RABV)-it causes deadly encephalitis in mammals. MOKV entry into host cells is mediated by its transmembrane glycoprotein G. First, G binds cellular receptors, triggering virion endocytosis. Then, in the acidic endosomal environment, G undergoes a conformational change from its pre- toward its post-fusion state that catalyzes the merger of the viral and endosomal membranes. Here, we have determined the crystal structure of a soluble MOKV G ectodomain in which the hydrophobic fusion loops have been replaced by more hydrophilic sequences. The crystal structure corresponds to a monomer that is similar to the protomer of the trimeric post-fusion state of vesicular stomatitis virus (VSV) G. However, by electron microscopy, we show that, at low pH, at the surface of pseudotyped VSV, MOKV spikes adopt the trimeric post-fusion conformation and have a tendency to reorganize into regular arrays. Sequence alignment between MOKV G and RABV G allows a precise location of RABV G antigenic sites. Repositioning MOKV G domains on VSV G pre-fusion structure reveals that antigenic sites are located in the most exposed part of the molecule in its pre-fusion conformation and are therefore very accessible to antibodies. Furthermore, the structure allows the identification of pH-sensitive molecular switches. Specifically, the long helix, which constitutes the core of the post-fusion trimer for class III fusion glycoproteins, contains many acidic residues located at the trimeric interface. Several of them, aligned along the helix, point toward the trimer axis. They have to be protonated for the post-fusion trimer to be stable. At high pH, when they are negatively charged, they destabilize the interface, which explains the conformational change reversibility. Finally, the present structure will be of great help to perform rational mutagenesis on lyssavirus glycoproteins.
莫科拉病毒（MOKV）属于狂犬病毒属。与其他属成员一样，包括狂犬病病毒（RABV），它会导致哺乳动物致命的脑炎。MOKV 进入宿主细胞是由其跨膜糖蛋白 G 介导的。首先，G 与细胞受体结合，触发病毒体的内吞作用。然后，在酸性内体环境中，G 发生从预融合状态到后融合状态的构象变化，催化病毒和内体膜的融合。在这里，我们确定了一个可溶性 MOKV G 外域的晶体结构，其中疏水性融合环已被更亲水的序列取代。该晶体结构对应于单体，类似于水疱性口炎病毒（VSV）G 的三聚体后融合状态的原体。然而，通过电子显微镜，我们表明，在低 pH 值下，在假型 VSV 的表面，MOKV 刺突采用三聚体后融合构象，并倾向于重新排列成规则的阵列。MOKV G 和 RABV G 之间的序列比对可以精确定位 RABV G 的抗原位点。将 MOKV G 结构域重新定位到 VSV G 前融合结构上，揭示了抗原位点位于分子前融合构象中最暴露的部分，因此非常容易被抗体识别。此外，该结构允许识别 pH 敏感的分子开关。具体来说，构成 III 类融合糖蛋白后融合三聚体核心的长螺旋含有许多位于三聚体界面的酸性残基。其中几个沿着螺旋排列，指向三聚体轴。它们必须被质子化，后融合三聚体才能稳定。在高 pH 值下，当它们带负电荷时，会破坏界面的稳定性，这解释了构象变化的可逆性。最后，目前的结构将极大地帮助对狂犬病毒糖蛋白进行合理的突变。
