Odongo Laura, Habtegebrael Betelihem H, Kiessling Volker, White Judith M, Tamm Lukas K
Center for Membrane and Cell Physiology, University of Virginia , Charlottesville, Virginia, USA.
Department of Molecular Physiology and Biological Physics, University of Virginia , Charlottesville, Virginia, USA.
Microbiol Spectr. 2023 Sep 20;11(5):e0190823. doi: 10.1128/spectrum.01908-23.
Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.
埃博拉病毒（EBOV）可引发出血热，病死率高达90%。EBOV的进入过程复杂，尚未完全明确。在附着于宿主细胞后，EBOV被转运至晚期内体/溶酶体，其糖蛋白（GP）在该处被加工成19 kDa的形式，该形式与EBOV细胞内受体尼曼-匹克C1型（Niemann-Pick type C1）结合。我们之前表明，组织蛋白酶抑制剂E-64d可阻断携带19 kDa GP的假病毒颗粒的感染，这表明需要进一步的组织蛋白酶作用来触发融合。然而，这一点尚未得到直接证实。由于19 kDa埃博拉GP融合发生在晚期内体中，我们设计了一个系统，其中利用富集的晚期内体来制备支持性平面内体膜（SPEMs），并通过全内反射荧光显微镜监测荧光（假）病毒颗粒的融合。我们通过证明流感病毒血凝素（HA）介导的和拉沙病毒（LASV）GP介导的融合对pH的依赖性来验证该系统。利用SPEMs，我们发现由19 kDa埃博拉GP介导的融合依赖于低pH，受Ca增强，并因添加组织蛋白酶而增强。随后，我们发现E-64d抑制由19 kDa GP介导的完全融合，但不抑制脂质混合，我们用可逆性组织蛋白酶抑制剂VBY-825证实了这一点。因此，我们提供了功能获得和功能丧失的证据，即进一步的组织蛋白酶作用增强了19 kDa埃博拉GP的融合活性。除了为埃博拉GP如何介导融合提供新的见解外，我们开发的采用SPEMs的方法现在可广泛用于研究病毒和毒素通过内体进入细胞的过程。重要性 埃博拉病毒是埃博拉病毒病的病原体，该病严重且往往致命。EBOV通过晚期内体/溶酶体进入细胞。病毒膜与内体膜融合之前的事件尚未完全明确。在本研究中，我们报告了一种研究病毒与内体膜融合的新系统。我们通过证明流感病毒和拉沙病毒融合对低pH的依赖性来验证该系统。此外，我们表明进一步的组织蛋白酶B作用增强了引发的埃博拉病毒糖蛋白的融合活性。最后，这种模型内体膜系统应有助于研究其他包膜病毒、非包膜病毒和酸激活细菌毒素的双层破坏机制。
