Structure-function studies of the tripartite Junin arenavirus GP-C

阿雷纳病毒在世界各地的啮齿动物种群中是地方性的，可以传播给人类导致急性出血热。缺乏预防性疫苗和有效的治疗方法，迫切需要解决公共卫生和生物防御方面的关切。因此，针对病毒包膜糖蛋白(GP-C)和病毒进入靶细胞的干预策略是有吸引力的。ArenaVirus GP-C的不同寻常之处在于，成熟的复合体保留了其切割信号肽(SSP)作为必需的亚基，与传统的受体结合蛋白(G1)和跨膜融合蛋白(G2)结合在一起。我们的初步结果表明，SSP与G2的胞外区相互作用，调节GP-C复合体的pH依赖的膜融合活性，并形成一类新发现的小分子融合抑制剂的靶点。SSP还与G2的细胞质结构域结合，调节GP-C复合体在细胞内的运输到细胞表面，以进行病毒粒子的组装和萌发。我们最近的证据表明，SSP和G2之间的细胞质相互作用是通过一个新的亚基间锌指基序来调节的。该项目的长期目标是确定促进病毒粒子组装和病毒进入的三方GP-C复合体中的结构-功能关系，以便在设计有效的抗病毒药物时利用GP-C的独特功能。为此，我们提出了三个具体的目标：1)详细描述SSP与G2胞外区的相互作用，并了解新发现的小分子ArenaVirus融合抑制剂的作用机制；2)确定连接SSP和G2细胞质结构域的一个不寻常的锌指基序中锌结合的分子需求，并表征其在GP-C复合体中的作用；3)研究SSP和亚单位间锌指在病毒形态发生中的作用。

Arenaviruses are endemic in rodent populations worldwide and can be transmitted to humans to cause acute hemorrhagic fevers. Prophylactic vaccines and effective therapies are lacking and urgently needed to address public health and biodefense concerns. Intervention strategies that target the viral envelope glycoprotein (GP-C) and virus entry into target cells are thus attractive. The arenavirus GP-C is unusual in that the mature complex retains its cleaved signal peptide (SSP) as an essential subunit in association with the conventional receptor-binding (G1) and transmembrane fusion (G2) proteins. Our preliminary results have shown that SSP interacts with the ectodomain of G2 to modulate pH-dependent membrane-fusion activity of the GP-C complex, and to form the target for a class of newly discovered small-molecule fusion inhibitors. SSP also associates with the cytoplasmic domain of G2 to regulate intracellular transport of the GP-C complex to the cell surface for virion assembly and budding. Our recent evidence indicates that the cytoplasmic interaction between SSP and G2 is mediated through a novel intersubunit zinc-finger motif. The broad, long-term goal of this project is to define the structure-function relationships in the tripartite GP-C complex that promote virion assembly and virus entry, in order to exploit the unique features of GP-C in the design of effective antiviral therapeutics. Towards this goal, we propose three specific aims: i) To characterize in molecular detail the interaction between SSP and the ectodomain of G2, and to understand the mechanism of action of the newly discovered small-molecule arenavirus fusion inhibitors; ii) To identify the molecular requirements for zinc binding in an unusual zinc-finger motif bridging SSP and the cytoplasmic domain of G2, and to characterize its role in the GP-C complex; and iii) To investigate the role of SSP and the intersubunit zinc finger in virion morphogenesis.