Entry of Human Coronavirus NL63 into the Cell
Entry of Human Coronavirus NL63 into the Cell
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DOI：
10.1128/jvi.01933-17
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发表时间：
2018-02-01
影响因子：
5.4
通讯作者：
Pyrc, Krzysztof
中科院分区：
文献类型：
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作者：
Milewska, Aleksandra;Nowak, Paulina;Pyrc, Krzysztof
人类冠状病毒NL 63（HCoV-NL 63）感染的第一步先前已有描述。该病毒通过使用硫酸乙酰肝素蛋白聚糖与靶细胞结合，并与ACE 2蛋白相互作用。随后的事件，包括病毒内化和贩运，仍有待阐明。在这项研究中，我们绘制了HCoV-NL 63进入LLC-Mk2细胞系和离体三维（3D）气管支气管组织的过程。使用各种技术，我们已经表明，HCoV-NL 63病毒粒子需要内吞作用才能成功进入LLC-MK2细胞，并且病毒与ACE 2分子之间的相互作用触发网格蛋白的募集。随后由发动蛋白引起的囊泡断裂导致病毒内化，并且新形成的囊泡通过肌动蛋白皮质，这需要主动的细胞骨架重排。最后，内体微环境的酸化是成功融合和释放病毒基因组进入细胞质所必需的。对于3D气管支气管组织培养，我们还观察到，病毒进入细胞网格蛋白介导的内吞作用，但我们得到的结果表明，这条途径可能是bypassed.IMPORTANCE可用的数据冠状病毒进入经常起源于研究采用永生化细胞系或未分化的细胞。在这里，使用最先进的3D组织培养系统模拟传导气道的上皮，我们系统地映射HCoV-NL 63进入易感细胞。所获得的数据可以更好地了解感染过程，并可能支持开发新的治疗策略。
The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into the LLC-Mk2 cell line and ex vivo three-dimensional (3D) tracheobronchial tissue. Using a variety of techniques, we have shown that HCoV-NL63 virions require endocytosis for successful entry into the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, which requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of the viral genome into the cytoplasm. For 3D tracheobronchial tissue cultures, we also observed that the virus enters the cell by clathrin-mediated endocytosis, but we obtained results suggesting that this pathway may be bypassed.IMPORTANCE Available data on coronavirus entry frequently originate from studies employing immortalized cell lines or undifferentiated cells. Here, using the most advanced 3D tissue culture system mimicking the epithelium of conductive airways, we systematically mapped HCoV-NL63 entry into susceptible cells. The data obtained allow for a better understanding of the infection process and may support development of novel treatment strategies.