Proteolytic cleavage of the Hendra virus fusion protein

描述（由申请人提供）：亨德拉病毒和密切相关的尼帕病毒是人畜共患副粘病毒，在人类中具有高死亡率，两者都已被列入NIAID优先病原体清单。亨德拉病毒的融合(F)蛋白促进病毒-细胞和细胞-细胞膜融合，从而介导病毒感染的关键第一步。这一重要的病毒蛋白经历了前体形式F0的蛋白水解裂解，裂解为二硫连接形式F1和F2，这一过程已被证明是迄今为止表征的所有副粘病毒F蛋白的融合活性所必需的。很明显，细胞分泌蛋白酶furin，已知可切割其他副粘病毒F蛋白，不参与Hendra F蛋白的蛋白水解激活。目前，这一关键裂解事件的细胞定位和所涉及的蛋白酶的身份都是未知的。这项拟议研究的长期目标是了解亨德拉F蛋白的加工过程，并确定参与这些事件的宿主细胞因子。需要验证的具体假设是，参与亨德拉F蛋白切割的蛋白酶代表了一类新的细胞蛋白酶，可以作为抗病毒治疗的靶标。在我们的初步结果中，我们证明蛋白水解激活发生在从反式高尔基网络出芽的分泌囊泡或细胞表面。此外，我们发现蛋白水解过程不受Ca2+水平的影响，但对细胞内ph的变化极为敏感。Aim 1中的实验将确定Hendra F蛋白蛋白水解过程的细胞位置以及切割所需的氨基酸序列。Aim 2中提出的研究旨在纯化负责Hendra F蛋白水解加工的蛋白酶，随后使用n端测序或蛋白质组学技术进行鉴定。这项研究将提供有关分泌途径基本过程的重要信息。此外，鉴定参与亨德拉和尼帕F蛋白蛋白水解裂解的酶将为开发针对这些最近出现的副粘病毒的新抗病毒疗法奠定基础。

DESCRIPTION (provided by applicant): Hendra virus and the closely related Nipah virus are zoonotic paramyxoviruses with high mortality rates in humans, and both have been placed on the NIAID priority pathogens list. The fusion (F) protein of Hendra virus promotes both virus-cell and cell-cell membrane fusion, thereby mediating a critical first step in viral infection. This important viral protein undergoes proteolytic cleavage of the precursor form, F0, to the disulfide-linked form, F1 and F2, a process that has been shown to be required for fusogenic activity of all paramyxovirus F proteins characterized to date. It is clear that the cellular secretory protease furin, known to cleave other paramxyovirus F proteins, is not involved in proteolytic activation of the Hendra F protein. At present, neither the cellular localization of this crucial cleavage event or the identity of the protease involved is known. The long-term objective of this proposed research is to understand the processing of the Hendra F protein, and to identify host cell factors involved in these events. The specific hypothesis to be tested is that the protease involved in cleavage of the Hendra F protein represents a novel class of cellular proteases that could serve as a target for anti-viral therapies. In our preliminary results, we demonstrate that proteolytic activation occurs in either secretory vesicles budding from the trans-Golgi network or at the cell surface. In addition, we find that proteolytic processing is not affected by Ca2+ levels, but is extremely sensitive to changes in intracellular pH. The experiments in Aim 1 will identify the cellular location of proteolytic processing of the Hendra F protein and the amino acid sequence requirements for cleavage. The research proposed in Aim 2 is directed towards purification of the protease responsible for proteolytic processing of the Hendra F protein, with subsequent identification using N-terminal sequencing or proteomic techniques. This research will provide important information about basic processes within the secretory pathway. In addition, identification of the enzyme(s) involved in the proteolytic cleavage of the Hendra and Nipah F proteins will set the stage for development of new antiviral therapies for these recently emerged paramyxoviruses.