PDF(Pubmed)
Abstract：
2019 coronavirus disease (COVID-19) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to respiratory illness, COVID-19 patients exhibit neurological symptoms lasting from weeks to months (long COVID). It is unclear whether these neurological manifestations are due to an infection of brain cells. We found that a small fraction of human induced pluripotent stem cell (iPSC)-derived neurons, but not astrocytes, were naturally susceptible to SARS-CoV-2. Based on the inhibitory effect of blocking antibodies, the infection seemed to depend on the receptor angiotensin-converting enzyme 2 (ACE2), despite very low levels of its expression in neurons. The presence of double-stranded RNA in the cytoplasm (the hallmark of viral replication), abundant synthesis of viral late genes localized throughout infected cells, and an increase in the level of viral RNA in the culture medium (viral release) within the first 48 h of infection suggested that the infection was productive. Productive entry of SARS-CoV-2 requires the fusion of the viral and cellular membranes, which results in the delivery of the viral genome into the cytoplasm of the target cell. The fusion is triggered by proteolytic cleavage of the viral surface spike protein, which can occur at the plasma membrane or from endosomes or lysosomes. We found that SARS-CoV-2 infection of human neurons was insensitive to nafamostat and camostat, which inhibit cellular serine proteases, including transmembrane serine protease 2 (TMPRSS2). Inhibition of cathepsin L also did not significantly block infection. In contrast, the neuronal infection was blocked by apilimod, an inhibitor of phosphatidyl-inositol 5 kinase (PIK5K), which regulates early to late endosome maturation. IMPORTANCE COVID-19 is a disease caused by the coronavirus SARS-CoV-2. Millions of patients display neurological symptoms, including headache, impairment of memory, seizures, and encephalopathy, as well as anatomical abnormalities, such as changes in brain morphology. SARS-CoV-2 infection of the human brain has been documented, but it is unclear whether the observed neurological symptoms are linked to direct brain infection. The mechanism of virus entry into neurons has also not been characterized. Here, we investigated SARS-CoV-2 infection by using a human iPSC-derived neural cell model and found that a small fraction of cortical-like neurons was naturally susceptible to infection. The productive infection was ACE2 dependent and TMPRSS2 independent. We also found that the virus used the late endosomal and lysosomal pathway for cell entry and that the infection could be blocked by apilimod, an inhibitor of cellular PIK5K.
摘要：
2019冠状病毒病（COVID-19）是由严重急性呼吸道综合症冠状病毒2（SARS-CoV-2）引起的疾病。除了呼吸道疾病,COVID-19患者表现出持续数周至数月的神经症状(长COVID)。尚不清楚这些神经系统表现是否是由于脑细胞感染所致。我们发现一小部分人诱导多能干细胞（iPSC）衍生的神经元，但不是星形胶质细胞，自然易患SARS-CoV-2。基于阻断抗体的抑制作用，感染似乎依赖于受体血管紧张素转换酶2（ACE2），尽管其在神经元中的表达水平非常低。细胞质中双链RNA的存在（病毒复制的标志），病毒晚期基因的丰富合成位于整个感染细胞，并且在感染的前48小时内培养基中病毒RNA水平的增加（病毒释放）表明感染是生产性的。SARS-CoV-2的生产性进入需要病毒和细胞膜的融合，这导致病毒基因组递送到靶细胞的细胞质中。融合是由病毒表面刺突蛋白的蛋白水解裂解引发的，它可以发生在质膜或从内体或溶酶体。我们发现SARS-CoV-2感染人类神经元对nafamostat和camostat不敏感，抑制细胞丝氨酸蛋白酶，包括跨膜丝氨酸蛋白酶2(TMPRSS2)。组织蛋白酶L的抑制也没有显著阻断感染。相比之下,神经元感染被阿吡莫德阻断，磷脂酰肌醇5激酶（PIK5K）的抑制剂，调节内体成熟的早期到晚期。重要性COVID-19是一种由冠状病毒SARS-CoV-2引起的疾病。数百万患者表现出神经症状，包括头痛,记忆受损，癫痫发作,和脑病，以及解剖异常，比如大脑形态的变化。SARS-CoV-2感染人脑已被证明，但目前尚不清楚观察到的神经症状是否与直接的脑部感染有关。病毒进入神经元的机制也没有被表征。这里,我们使用人类iPSC来源的神经细胞模型研究了SARS-CoV-2感染,发现一小部分皮质样神经元天然易感感染.生产性感染是ACE2依赖性和TMPRSS2依赖性。我们还发现该病毒使用晚期内体和溶酶体途径进入细胞，并且感染可以被阿吡莫德阻断，细胞PIK5K的抑制剂。
