Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA.
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA
J Virol. 2019 Feb 5;93(4). doi: 10.1128/JVI.01881-18. Print 2019 Feb 15.
Alphaviruses are small enveloped RNA viruses that bud from the host cell plasma membrane. Alphavirus particles have a highly organized structure, with a nucleocapsid core containing the RNA genome surrounded by the capsid protein, and a viral envelope containing 80 spikes, each a trimer of heterodimers of the E1 and E2 glycoproteins. The capsid protein and envelope proteins are both arranged in organized lattices that are linked via the interaction of the E2 cytoplasmic tail/endodomain with the capsid protein. We previously characterized the role of two highly conserved histidine residues, H348 and H352, located in an external, juxtamembrane region of the E2 protein termed the D-loop. Alanine substitutions of H348 and H352 inhibit virus growth by impairing late steps in the assembly/budding of virus particles at the plasma membrane. To investigate this budding defect, we selected for revertants of the E2-H348/352A double mutant. We identified eleven second-site revertants with improved virus growth and mutations in the capsid, E2 and E1 proteins. Multiple isolates contained the mutation E2-T402K in the E2 endodomain or E1-T317I in the E1 ectodomain. Both of these mutations were shown to partially restore H348/352A growth and virus assembly/budding, while neither rescued the decreased thermostability of H348/352A. Within the alphavirus particle, these mutations are positioned to affect the E2-capsid interaction or the E1-mediated intertrimer interactions at the 5-fold axis of symmetry. Together, our results support a model in which the E2 D-loop promotes the formation of the glycoprotein lattice and its interactions with the internal capsid protein lattice. Alphaviruses include important human pathogens such as Chikungunya and the encephalitic alphaviruses. There are currently no licensed alphavirus vaccines or effective antiviral therapies, and more molecular information on virus particle structure and function is needed. Here, we highlight the important role of the E2 juxtamembrane D-loop in mediating virus budding and particle production. Our results demonstrated that this E2 region affects both the formation of the external glycoprotein lattice and its interactions with the internal capsid protein shell.
甲病毒是从小型包膜 RNA 病毒中芽生的，从宿主细胞质膜中芽生。甲病毒颗粒具有高度组织化的结构，核衣壳核心包含 RNA 基因组，周围是衣壳蛋白，病毒包膜包含 80 个刺突，每个刺突都是 E1 和 E2 糖蛋白异二聚体的三聚体。衣壳蛋白和包膜蛋白都以有组织的晶格排列，通过 E2 细胞质尾巴/内域与衣壳蛋白的相互作用连接。我们之前描述了位于 E2 蛋白的外部、近膜区的两个高度保守的组氨酸残基 H348 和 H352 的作用，该区域称为 D 环。H348 和 H352 的丙氨酸取代通过损害包膜蛋白在质膜上的晚期组装/芽生步骤抑制病毒生长。为了研究这种出芽缺陷，我们选择了 E2-H348/352A 双突变体的回复突变体。我们鉴定了 11 个具有改善的病毒生长能力的第二位置回复突变体，并且在衣壳、E2 和 E1 蛋白中发生突变。多个分离株含有 E2 内域中的 E2-T402K 或 E1 外域中的 E1-T317I 突变。这两个突变都部分恢复了 H348/352A 的生长和病毒组装/出芽，而没有挽救 H348/352A 的降低的热稳定性。在甲病毒颗粒内，这些突变的位置会影响 E2-衣壳相互作用或 5 倍对称轴处的 E1 介导的三聚体相互作用。总之，我们的结果支持这样一种模型，即 E2 D 环促进糖蛋白晶格的形成及其与内部衣壳蛋白晶格的相互作用。甲病毒包括重要的人类病原体，如基孔肯雅热病毒和脑炎甲病毒。目前没有许可的甲病毒疫苗或有效的抗病毒疗法，需要更多关于病毒颗粒结构和功能的分子信息。在这里，我们强调了 E2 近膜 D 环在介导病毒出芽和颗粒产生中的重要作用。我们的结果表明，该 E2 区域影响外部糖蛋白晶格的形成及其与内部衣壳蛋白壳的相互作用。
