Ebola Virus and Severe Acute Respiratory Syndrome Coronavirus Display Late Cell Entry Kinetics: Evidence that Transport to NPC1+ Endolysosomes Is a Rate-Defining Step
Ebola Virus and Severe Acute Respiratory Syndrome Coronavirus Display Late Cell Entry Kinetics: Evidence that Transport to NPC1+ Endolysosomes Is a Rate-Defining Step
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DOI：
10.1128/jvi.03398-14
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发表时间：
2015-03-01
影响因子：
5.4
通讯作者：
White, Judith M.
中科院分区：
文献类型：
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作者：
Mingo, Rebecca M.;Simmons, James A.;White, Judith M.
埃博拉病毒（EBOV）引起出血热，死亡率很高。在进入细胞过程中，病毒通过巨噬细胞作用内化并通过内体运输，直到触发病毒与内体膜融合，将RNA基因组释放到细胞质中。我们发现，虽然表达EBOV糖蛋白（GP）的丝状EBOV病毒样颗粒（VLPs）的巨噬细胞摄取相对较快，但VLPs在30分钟后才开始进入细胞质，远远晚于携带来自淋巴细胞脉络丛脑膜炎病毒的流感血凝素或GP的颗粒，后者通过晚期内体（LE）进入。对于EBOV来说，长时间的延迟不是由于EBOV丝的大尺寸或不寻常的形状，需要将EBOV GP引到19 kda受体结合种，或者需要异常低的内体ph。相反，由于我们观察到EBOV在到达neemann - pick C1 （NPC1）阳性内溶酶体（LE/Lys）时发生进入，我们认为运输到LE/Lys是一个关键的速率决定步骤。另外的实验出乎意料地显示，严重急性呼吸综合征（SARS） s介导的进入也只在30分钟后才开始。此外，尽管SARS不需要NPC1进入，但SARS也在与NPC1共域后开始进入。由于SARS进入内体的唯一要求是组织蛋白酶L活性，我们测试并提供证据表明NPC1(+) LE/Lys比LE具有更高的组织蛋白酶L活性，而在早期内体中没有检测到活性。我们的研究结果表明，EBOV和SARS都深入内吞途径进入，并且它们这样做是为了获得更高的组织蛋白酶活性。埃博拉病毒是一种出血热病毒，当它从人畜共患病媒介传播到人群中时，会导致高死亡率。感染严重急性呼吸综合征冠状病毒（SARS-CoV）的患者会出现严重的呼吸窘迫。2014年西非爆发了一场毁灭性的埃博拉病毒疫情，2003年又爆发了严重的SARS疫情。目前还没有针对这两种病毒的有效疫苗或治疗方法被批准。我们提供的证据表明，这两种病毒都很晚才进入内吞途径，到达NPC1(+) LE/Lys，以便进入宿主细胞，并且它们这样做是为了获得高水平的组织蛋白酶活性，这两种病毒在其融合触发机制中都使用了组织蛋白酶活性。这种意想不到的相似性表明，NPC1(+) LE/Lys作为SARS和EBOV的治疗靶点存在一种未被发现的脆弱性。
Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity.IMPORTANCEEbola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV.