Elucidating the Mechanism of Phosphatidylserine Exposure During Ebola Virus Assembly.
Elucidating the Mechanism of Phosphatidylserine Exposure During Ebola Virus Assembly.
复制标题
阐明埃博拉病毒组装过程中磷脂酰丝氨酸暴露的机制。
DOI：
10.1093/micmic/ozad067.461
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Wan,William
中科院分区：
文献类型：
--
作者：
Huth,Tyler;Wan,William
埃博拉病毒（EBOV）是一种有包膜的负义单链RNA病毒，在中非和西非持续存在，是一种新出现的威胁，暴发记录的病死率高达90%。与EBOV感染相关的高致死率通常与其广泛的细胞嗜性相关，有效地允许在几乎任何组织中进入和复制。这种广泛的嗜性被认为是由于EBOV附着和进入细胞的非特异性性质。在已知的附着因子中，有T细胞免疫球蛋白和粘蛋白结构域（TIM）家族和受体酪氨酸激酶Tyro3、Axl和Mer的TAM家族的成员。已知TIM和TAM受体家族都与磷脂酰丝氨酸（PtdSer）结合。PtdSer主要富集在健康细胞质膜的细胞内小叶上，但这种不对称性的维持在细胞凋亡期间被破坏以暴露细胞表面上的脂质。外化PtdSer作为“吃我”信号传递给邻近的吞噬细胞，以促进凋亡细胞碎片的清除。某些病毒，包括EBOV，采用称为凋亡模拟的策略，其中病毒体在其宿主来源的病毒包膜上展示PtdSer，以通过凋亡清除途径进入细胞。虽然有证据表明EBOV在进入靶细胞期间采用凋亡模拟，但关于病毒在病毒组装期间如何破坏PtdSer的不对称分布知之甚少。在EBOV生命周期期间，EBOV从质膜组装和出芽。EBOV基质蛋白VP40在质膜的细胞内表面上寡聚化并协调病毒组装和出芽。EBOV的糖蛋白（GP）在受体结合和膜融合中起关键作用。单独表达VP40足以产生丝状病毒样颗粒（VLP），并且在与GP共转染后可以增强相对出芽效率。此外，已经证明单独的VP40表达诱导PtdSer的暴露。研究已经注意到，在与GP共转染时实现PtdSer的更稳健暴露，表明GP表达以协调的方式促进出芽和PtdSer外化。VP40和GP表达触发PtdSer暴露的机制知之甚少。为了确定VP40和GP在病毒组装过程中的时空动力学，我们正在开发一种可诱导的活细胞成像系统来监测病毒蛋白浓度、亚细胞定位和真实的PtdSer暴露。此外，我们试图解决结构中间体的VP40和GP组件的质膜上使用冷冻电子断层扫描表征病毒蛋白与膜的相互作用在分子尺度上。总之，活细胞显微镜和EBOV的VP40和GP的结构表征将阐明病毒蛋白表达诱导PtdSer暴露的机制。
Ebola virus (EBOV) is an enveloped, negative sense, single-stranded RNA virus which persists as an emerging threat in Central and Western Africa with outbreaks recording case fatality rates as high as 90%. The high fatality rates associated with EBOV infection are often correlated with its broad cell tropism, effectively allowing for entry and replication in almost any tissue. This broad tropism is thought to be due to the nonspecific nature by which EBOV attaches to and enters cells. Among the known attachment factors are members of the T-cell immunoglobulin and mucin domain (TIM) family and the TAM family of receptor tyrosine kinases, Tyro3, Axl, and Mer. Both the TIM and TAM family of receptors are known to engage with the phospholipid phosphatidylserine (PtdSer). PtdSer is predominantly enriched on the intracellular leaflet of healthy cell plasma membranes, but maintenance of this asymmetry is disrupted during apoptosis to expose the lipid on the cell’s surface. Externalized PtdSer serves as an “eat me” signal to neighboring phagocytes to promote the clearance of apoptotic cell debris. Certain viruses, including EBOV, employ a strategy known as apoptotic mimicry where virions display PtdSer on their host-derived viral envelopes to enter cells via the apoptotic clearance pathways. While evidence indicates EBOV’s employment of apoptotic mimicry during entry into target cells, little is known regarding how the virus disrupts PtdSer’s asymmetric distribution during viral assembly. During the EBOV life cycle, EBOV assembles and buds from the plasma membrane. The EBOV matrix protein, VP40, oligomerizes on the intracellular face of the plasma membrane and coordinates viral assembly and budding. The glycoprotein (GP) of EBOV plays a critical role in receptor binding and membrane fusion. Expression of VP40 alone is sufficient for generation of filamentous virus-like particles (VLPs) and the relative budding efficiency can be enhanced upon co-transfection with GP. Additionally, it has been demonstrated that VP40 expression alone induces exposure of PtdSer. Studies have noted that more robust exposure of PtdSer is achieved upon co-transfection with GP suggesting that GP expression promotes budding and PtdSer externalization in a coordinated manner. The mechanism by which VP40 and GP expression triggers exposure of PtdSer is poorly understood. To determine the spatiotemporal dynamics of both VP40 and GP during viral assembly, we are developing an inducible live-cell imaging system to monitor viral protein concentration, sub cellular localization, and PtdSer exposure in real time. Furthermore, we seek to resolve structural intermediates of VP40 and GP assemblies on the plasma membrane using cryo-electron tomography to characterize viral protein interactions with the membrane at the molecular scale. Together, live-cell microscopy and structural characterization of EBOV’s VP40 and GP will elucidate a mechanism by which viral protein expression induces PtdSer exposure.