Proteolytic cleavage of the Hendra virus fusion protein

性状（由申请方提供）：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，均已被列入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，从而介导病毒感染中关键的第一步。这种重要的病毒蛋白经历前体形式F0的蛋白水解裂解，成为二硫键连接形式F1和F2，这一过程已被证明是迄今为止所有副粘病毒F蛋白的融合活性所必需的。很明显，已知切割其他副腺病毒F蛋白的细胞分泌蛋白酶弗林蛋白酶不参与亨德拉F蛋白的蛋白水解活化。目前，这一关键的裂解事件的细胞定位或所涉及的蛋白酶的身份都是未知的。这项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。待检验的具体假设是，参与Hendra F蛋白裂解的蛋白酶代表一类新型细胞蛋白酶，可用作抗病毒治疗的靶标。在我们的初步研究结果中，我们证明，蛋白水解激活发生在分泌囊泡萌芽从trans-Golgi网络或在细胞表面。此外，我们发现，蛋白水解加工不受Ca 2+水平的影响，但在细胞内pH值的变化是非常敏感的。目的1的实验将确定亨德拉F蛋白的蛋白水解加工的细胞位置和切割的氨基酸序列要求。目标2中提出的研究针对负责亨德拉F蛋白的蛋白水解加工的蛋白酶的纯化，随后使用N-末端测序或蛋白质组学技术进行鉴定。这项研究将提供重要的信息，分泌途径内的基本过程。此外，参与Hendra和Nipah F蛋白的蛋白水解裂解的酶的鉴定将为开发针对这些最近出现的副粘病毒的新的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.