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中华实验和临床感染病杂志(电子版)

中华实验和临床感染病杂志(电子版) ›› 2019, Vol. 13 ›› Issue (05) : 370 -376. doi: 10.3877/cma.j.issn.1674-1358.2019.05.005

所属专题： 文献；

论著

蝙蝠来源的重症急性呼吸综合征样冠状病毒WIV1刺突蛋白利用浣熊狗血管紧张素转换酶Ⅱ受体侵入细胞能力的研究
郑梅1, 郑双丽1, 陈丹瑛1, 蒋栋1, 曾辉1, 赵学森1,†()   
  1. 1. 100015　北京，首都医科大学附属北京地坛医院传染病研究所
  • 收稿日期:2019-04-08 出版日期:2019-10-15
  • 通信作者: 赵学森
  • 基金资助:
    国家自然科学基金(No. 81571976)

Ability of bat severe acute respiratory syndromes-like coronavirus (WIV1) to utilize raccoon dog angiotensin-converting enzyme as receptor for cellular entry

Mei Zheng1, Shuangli Zheng1, Danying Chen1, Dong Jiang1, Hui Zeng1, Xuesen Zhao1,†()   

  1. 1. Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
  • Received:2019-04-08 Published:2019-10-15
  • Corresponding author: Xuesen Zhao
  • About author:
    Corresponding author: Zhao Xuesen, Email:
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郑梅, 郑双丽, 陈丹瑛, 蒋栋, 曾辉, 赵学森. 蝙蝠来源的重症急性呼吸综合征样冠状病毒WIV1刺突蛋白利用浣熊狗血管紧张素转换酶Ⅱ受体侵入细胞能力的研究[J]. 中华实验和临床感染病杂志(电子版), 2019, 13(05): 370-376.

Mei Zheng, Shuangli Zheng, Danying Chen, Dong Jiang, Hui Zeng, Xuesen Zhao. Ability of bat severe acute respiratory syndromes-like coronavirus (WIV1) to utilize raccoon dog angiotensin-converting enzyme as receptor for cellular entry[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2019, 13(05): 370-376.

目的

研究蝙蝠来源的重症急性呼吸综合征（SARS）样冠状病毒WIV1刺突蛋白利用浣熊狗血管紧张素转换酶Ⅱ（ACE2）受体侵入细胞的能力，并探究SARS样冠状病毒WIV1潜在的跨宿主传播风险。

方法

构建来自浣熊狗、果子狸、中华菊头蝠和人等不同动物来源的ACE2表达质粒，转染至293T细胞并利用免疫印迹方法检测其在293T细胞中的表达水平；建立SARS冠状病毒（Tor2株系）及蝙蝠SARS样冠状病毒（WIV1株系）假病毒感染系统，并进行假病毒感染实验；利用假病毒感染系统及荧光素酶报告基因检测WIV1刺突蛋白对浣熊狗等不同动物来源ACE2受体利用能力；构建跨膜丝氨酸蛋白酶2（TMPRSS2）质粒，并转染至T Rex 293细胞，借助假病毒感染系统检测TMPRSS2对WIV1侵入能力的影响。

结果

本研究所获赠及构建的不同动物来源的ACE2质粒可经瞬时转染至细胞进行表达；与pcDNA3.1载体相比，浣熊狗、中华菊头蝠、果子狸和人的ACE2均可使蝙蝠SARS样冠状病毒WIV1侵入细胞的能力增加上万倍，上述ACE2组与载体组的荧光素酶活性差异均具有统计学意义（t = 27.744、P < 0.001，t = 18.740、P < 0.001，t = 32.297、P < 0.001，t = 15.902、P < 0.001）；与TMPRSS2阴性组相比，TMPRSS2在靶细胞的表达可使WIV1假病毒的感染能力增加10倍以上，两组荧光素酶活性差异具有统计学意义（t = 29.460、P < 0.001）。

结论

蝙蝠SARS样冠状病毒WIV1的刺突蛋白除了利用人类、果子狸及中华菊头蝠的ACE2受体感染细胞，还可利用浣熊狗的ACE2受体侵入细胞，且TMPRSS2可显著促进其侵入能力，提示WIV1可能存在多种跨宿主传播的风险。

关键词: 蝙蝠，重症急性呼吸综合征样冠状病毒, WIV1, 浣熊狗, 人类血管紧张素转换酶Ⅱ, 跨膜丝氨酸蛋白酶2
Objective

To investigate the ability of bat severe acute respiratory syndromes (SARS)-like coronavirus (WIV1 strain) to utilize angiotensin-converting enzyme Ⅱ (ACE2) from raccoon dog as a receptor for cellular entry, and to explore the potential cross-species transmissibility of WIV1.

Methods

Plasmids encoding ACE2 molecules from different animals including human (Homo sapiens), civet cat (Paguma larvata), Chinese horseshoe bat (Rhinolophus sinicus) and raccoon dog (Nyctereutes procyonoides) were constructed, and then transfected into 293T cells. The expressions of ACE2 proteins in 293T were detected by Western blot with anti-C9 antibody. The pseudoviral infection systems of SARS coronavirus (Tor2 strain) and bat SARS-like coronavirus (WIV1 strain) were established to detect receptor activity of different animals’ ACE2 for WIV1 entry. The plasmid expressing transmembrane serine protease 2 (TMPRSS2) was constructed, and then transfected into T Rex 293 cells. The effect of TMPRSS2 on WIV1pp infectivity was examined by luciferase assay.

Results

ACE2 from raccoon dog, Chinese horseshoe bat, civet cat and human expressed well in 293T cells and supported cellular entry mediated by WIV1 S protein. Compared with pcDNA3.1 vector, ACE2 molecules from raccoon dog, Chinese horseshoe bat, civet cat and human increased the WIV1pp infection by thousands of times, all with significant differences in luciferase activity (t = 27.744, P < 0.001; t = 18.740, P < 0.001; t = 32.297, P < 0.001; t = 15.902, P < 0.001 ). Compared with the group without TMPRSS2, the expression of TMPRSS2 in target cells increased the infection mediated by WIV1-S protein by more than 10 folds, with significant difference in luciferase activity (t = 29.460, P < 0.001).

Conclusions

The ACE2 molecules from raccoon dog served as a functional receptor for cellular entry mediated by WIV1 spike protein, which was activated by the TMPRSS2 protease. Our findings herein suggest that there may exist a risk of multiple cross-species transmission of WIV1 among human, bat and raccoon dog.

Key words: Bat severe acute respiratory syndromes-like coronavirus, WIV1, Raccoon dog, Angiotensin-converting enzyme Ⅱ, Transmembrane protease serine 2
图1 ACE2蛋白表达及其介导的细胞融合
图2 SARS-CoV（Tor2株系）假病毒和SARS样冠状病毒WIV1假病毒利用不同动物来源ACE2受体侵入细胞的能力
图3 TMPRSS2对SARS-CoV（Tor2株系）假病毒及蝙蝠SARS样冠状病毒WIV1假病毒感染能力的影响
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