埃博拉病毒(EBOV)导致出血热,致死率高达90%。EBOV进入过程复杂且不完全理解。附着于宿主细胞后,EBOV被运输到晚期内体/溶酶体,其糖蛋白(GP)被加工成19kDa的形式,它与EBOV细胞内受体Niemann-PickC1型结合。我们以前证明了组织蛋白酶抑制剂,E-64d,阻断携带19kDaGP的假病毒颗粒感染,提示需要进一步的组织蛋白酶作用来触发融合.这个,然而,没有直接证明。由于19kDa埃博拉GP融合发生在晚期内体,我们设计了一个系统,其中富集的晚期内体用于制备支持的平面内体膜(SPEMs),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介导的融合的pH依赖性来验证系统。使用SPEM,我们表明,由19kDa埃博拉GP介导的融合依赖于低pH,由Ca2+增强,并通过添加组织蛋白酶来增强。随后,我们发现E-64d抑制完全融合,但不是脂质混合,由19kDaGP介导,我们用可逆的组织蛋白酶抑制剂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,表明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.除了提供有关埃博拉GP如何介导融合的新见解外,我们开发的采用SPEMs的方法现在可以广泛用于病毒和毒素通过内体进入的研究.重要性埃博拉病毒是埃博拉病毒病的病原体,这是严重的,往往是致命的。EBOV通过晚期内体/溶酶体进入细胞。病毒和内体膜融合之前的事件尚未完全理解。在这项研究中,我们报道了一种新的体外系统,用于研究病毒与内体膜的融合。我们通过证明流感和拉沙病毒融合的低pH依赖性来验证该系统。此外,我们显示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,该模型内体膜系统应该有助于研究其他包膜病毒破坏双层的机制,通过无包膜病毒,和酸激活的细菌毒素。
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 Ca2+, 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 in vitro 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.