巨胞饮途径介导的新冠原始株病毒蛋白入胞的细胞水平初步研究

doi:10.3969/j.issn.1674-8115.2022.08.003

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (8): 987-996.doi: 10.3969/j.issn.1674-8115.2022.08.003

巨胞饮途径介导的新冠原始株病毒蛋白入胞的细胞水平初步研究

江淦¹(), 杨于权², 陈曜星¹, 侯照远², 高小玲¹, 陈红专¹^,³(), 贾浩²()

^1.上海交通大学基础医学院药理学与化学生物学系，上海高校转化医学协同创新中心，上海 200025
^2.上海交通大学基础医学院生物化学与分子细胞生物学系，上海市肿瘤微环境与炎症重点实验室，上海 200025
^3.上海中医药大学交叉科学研究院，上海 201203

收稿日期:2022-03-16 接受日期:2022-05-18 出版日期:2022-06-29 发布日期:2022-06-29
通讯作者: 陈红专,贾浩 E-mail:scmcyidingkaixin@163.com;hongzhuan_chen@hotmail.com;fonney@sjtu.edu.cn
作者简介:江淦（1987—），男，副教授，博士；电子信箱：scmcyidingkaixin@163.com。
基金资助:
国家自然科学基金(82171358);上海市青年科技启明星计划(19QA1405000);上海市高水平地方高校创新团队(SHSMU-ZDCX20211801)

Preliminary study on the cellular level of SARS-CoV-2 proteins mediated by macropinocytosis pathway

JIANG Gan¹(), YANG Yuquan², CHEN Yaoxing¹, HOU Zhaoyuan², GAO Xiaoling¹, CHEN Hongzhuan¹^,³(), JIA Hao²()

^1.Shanghai Universities Collaborative Innovation Center for Translational Medicine, Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Department of Biochemistry & Molecular Cellular Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^3.Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Received:2022-03-16 Accepted:2022-05-18 Online:2022-06-29 Published:2022-06-29
Contact: CHEN Hongzhuan,JIA Hao E-mail:scmcyidingkaixin@163.com;hongzhuan_chen@hotmail.com;fonney@sjtu.edu.cn
Supported by:
National Natural Science Foundation of China(82171358);Shanghai Rising-Star Program(19QA1405000);Innovative Research Team of High-level Local Universities in Shanghai(SHSMU-ZDCX20211801)

摘要/Abstract

摘要：

目的·探讨严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）原始株的几种关键蛋白对多种细胞模型巨胞饮途径的影响。方法·①利用免疫共沉淀技术探索SARS-COV-2原始株的刺突蛋白受体结合域（spike protein receptor-binding domain，S-RBD）、核衣壳蛋白（nucleocapsid protein，N）和非结构蛋白7（non-structural protein-7，NSP7）等病毒蛋白与HEK-293T细胞内蛋白的相互作用。②于体外，将SARS-CoV-2原始株的S-RBD、N、NSP7病毒蛋白分别与HEK-293T/bEnd.3/Beas-2b细胞（正常细胞模型）共孵育，利用巨胞饮标志物——异硫氰酸荧光素（fluorescein isothiocyanate，FITC）标记的70 kDa葡聚糖（FITC-70 kDa-葡聚糖）水平的变化观察上述细胞巨胞饮水平的改变。③利用脂多糖（lipopolysaccharide，LPS）诱导的炎症细胞模型，分析SARS-CoV-2原始株的病毒蛋白对该炎症细胞的巨胞饮水平的改变。④在正常细胞模型和炎症细胞模型中，利用5-（N-乙基-N-异丙基）阿米洛利（EIPA）或载带Rab5小干扰RNA（small interfering RNA，siRNA）的脂蛋白纳米药物载体分别抑制由SARS-CoV-2病毒蛋白诱导的巨胞饮作用，进一步观察细胞对S-RBD、N、NSP7病毒蛋白的摄取情况。结果·① SARS-CoV-2原始株的3个病毒蛋白在被摄取入胞后，可与Rab蛋白家族发生结合。②研究发现，SARS-CoV-2原始株的S-RBD、N、NSP7病毒蛋白均可刺激HEK-293T/bEnd.3/Beas-2b细胞产生巨胞饮作用。③在炎症细胞模型中，3个病毒蛋白均可增强细胞的巨胞饮作用。④经EIPA（75 μmol/L）或载带Rab5 siRNA的脂蛋白纳米药物载体处理后，2类细胞对S-RBD、N、NSP7病毒蛋白的摄取均有减少。结论·SARS-CoV-2原始株的S-RBD、N、NSP7病毒蛋白可上调多种细胞模型的巨胞饮水平，尤其是在合并炎症感染的情况下；同时，巨胞饮抑制剂/脂蛋白纳米药物载体均可抑制由上述病毒蛋白上调的巨胞饮作用，继而减少病毒蛋白的入胞水平。

关键词: 巨胞饮, 严重急性呼吸综合征冠状病毒2, 5-（N-乙基-N-异丙基）阿米洛利, Rab5

Abstract:

Objective ·To investigate the effects of several key proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on macropinocytosis in various cell models. Methods ·① The interactions between spike protein receptor-binding domain (S-RBD), nucleocapsid protein (N) and non-structural protein-7 (NSP7) of SARS-COV-2 and HEK-293T intracellular proteins were explored by co-immunoprecipitation assay. ② In vitro, S-RBD, N and NSP7 proteins of SARS-CoV-2 were incubated with HEK-293T/bEnd.3/Beas-2b cells (normal cell models), respectively, and the changes of macropinocytosis level of cells labeled with fluorescein isothiocyanate (FITC)-70 kDa-dextran were observed. ③ In vitro, S-RBD, N and NSP7 proteins of SARS-CoV-2 were incubated with inflammatory cells induced by lipopolysaccharide (LPS), respectively, and the changes of macropinocytosis level of inflammatory cells were analyzed. ④ In the normal cell models and inflammatory cell model, EIPA or lipoprotein nano-drug carriers loaded with Rab5 small interfering RNA (siRNA) were used to inhibit the macropinocytosis induced by SARS-CoV-2 proteins, respectively, and the uptake of S-RBD, N and NSP7 proteins by cells were further observed. Results ·① The three proteins of SARS-COV-2 could bind to Rab small GTPase proteins after being absorbed into cells. ② It was found that S-RBD, N and NSP7 proteins of SARS-COV-2 could induce the macropinocytosis after entering the HEK-293T/bEnd.3/Beas-2b cells. ③ Furthermore, the three proteins of SARS-COV-2 could enhance the megapinocytosis of the inflammatory cell. ④ After treatment with EIPA (75 μmol/L) or lipoprotein nano-drug carriers loaded with Rab5 siRNA, the uptake of S-RBD, N and NSP7 proteins were decreased in both types of cells. Conclusion ·S-RBD, N and NSP7 proteins of SARS-CoV-2 can up-regulate megapinocytosis levels in various cell models, especially in the case of combined inflammation infection. At the same time, macropinocytosis inhibitor / lipoprotein nano-drug carrier can inhibit the macropinocytosis up-regulated by the above proteins, and then reduce the entry levels of viral proteins.

Key words: macropinocytosis, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), EIPA, Rab5

中图分类号:

R373.1

江淦, 杨于权, 陈曜星, 侯照远, 高小玲, 陈红专, 贾浩. 巨胞饮途径介导的新冠原始株病毒蛋白入胞的细胞水平初步研究[J]. 上海交通大学学报（医学版）, 2022, 42(8): 987-996.

JIANG Gan, YANG Yuquan, CHEN Yaoxing, HOU Zhaoyuan, GAO Xiaoling, CHEN Hongzhuan, JIA Hao. Preliminary study on the cellular level of SARS-CoV-2 proteins mediated by macropinocytosis pathway[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 987-996.

图/表 8

表 1 Rab5 siRNA序列

Tab 1 Sequences of Rab5 siRNA

siRNA	Forward sequence （5'→3'）	Reverse sequence （5'→3'）
siRab5-1	AAGUACUAGUUUGAACUGGTT	CCAGUUCAAACUAGUACUUTT
siRab5-2	AUAGGACUGUGCUUCCUGGTT	CCAGGAAGCACAGUCCUAUTT
siRab5-3	UUACUACAACACUGAUUCCTT	GGAAUCAGUGUUGUAGUAATT

图1 Western blotting检测His-S-RBD、His-N和His-NSP7蛋白的纯化表达

Fig 1 Detection of purified expressions of His-S-RBD, His-N and His-NSP7 proteins by Western blotting

图2 SARS-COV-2原始株的S-RBD、N和NSP7蛋白与Rab5小GTPase家族蛋白的相互作用Note：A. Analysis of His-S-RBD, His-N and His-NSP7 binding proteins by co-immunoprecipitation. B. Venn diagram of differential proteins binding to S-RBD, N and NSP7 proteins in Rab small GTPase family. C. Heat map analysis of Rab small GTPase family binding to S-RBD, N and NSP7 proteins, respectively. D. Detection of the interaction of exogenous S-RBD, N and NSP7 proteins with endogenous Rab5 by co-immunoprecipitation and Western blotting, respectively. The S-RBD, N and NSP7 proteins and IgG (5 μg/mL) were co-cultured with HEK-293T cells for 3 h, respectively, and co-immunoprecipitation was performed with His-beads. E. Detection of the interaction of endogenous Rab5 with exogenous S-RBD, N and NSP7 proteins by co-immunoprecipitation and Western blotting, respectively. The S-RBD, N and NSP7 proteins and Vector (5 μg/mL) were co-cultured with HEK-293T cells for 3 h, respectively, and co-immunoprecipitation was performed with protein A/G beads.

Fig 2 Interaction between S-RBD, N and NSP7 proteins of SARS-COV-2 and Rab5 small GTPase family proteins

图 3 SARS-COV-2原始株的S-RBD、N和NSP7蛋白刺激HEK-293T细胞的巨胞饮作用Note：A/B. Analysis of the mean fluorescence intensity (A) and fluorescence image (B) of HEK-293T cells internalized with FITC-70 kDa-dextran by high content screening, after incubation with different concentrations of viral proteins for different times (×10). C. Co-localization of viral proteins and FITC-70 kDa-dextran in cells by confocal microscopy (×40). Scale bar=10 μm. D. Pearson's correlation analysis of viral proteins co-localization to FITC-70 kDa-dextran. E. Analysis of the fluorescence level of cells internalized with FITC-70 kDa-dextran by flow cytometry, after incubation with three viral proteins (5 μg/mL) for 3 h. *P<0.05.

Fig 3 S-RBD, N and NSP7 proteins of SARS-COV-2 stimulated the macropinocytosis in HEK-293T cells

图 4 SARS-COV-2原始株的S-RBD、N和NSP7蛋白刺激bEnd.3和Beas-2b细胞的巨胞饮作用Note：A/B. Analysis of the mean fluorescence intensity of bEnd.3 cells (A) and Beas-2b cells (B) internalized with FITC-70 kDa-dextran by high content screening, after incubation with different concentrations of viral proteins for different times. C/D. Analysis of the fluorescence image of bEnd.3 cells (C) and Beas-2b cells (D) internalized with FITC-70 kDa-dextran by high content screening, after incubation with different concentrations of viral proteins for different times (×10). E/F. Analysis of the fluorescence level of bEnd.3 cells (E) and Beas-2b cells (F) internalized with FITC-70 kDa-dextran by flow cytometry, after incubation with three viral proteins (5 μg/mL) for 3 h. *P<0.05.

Fig 4 S-RBD, N and NSP7 proteins of SARS-COV-2 stimulated the macropinocytosis in bEnd.3 and Beas-2b cells

图 5 SARS-CoV-2原始株的S-RBD、N和NSP-7蛋白刺激LPS诱导的炎症细胞的巨胞饮作用Note：A. Co-localization of viral proteins and FITC-70 kDa-dextran in LPS-induced HEK-293T cells by confocal microscopy (×40). Scale bar=10 μm. B. Pearson's correlation analysis of viral proteins co-localization to FITC-70 kDa-dextran in LPS-induced HEK-293T cells. C. Analysis of the fluorescence level of LPS-induced HEK-293T cells internalized with FITC-70 kDa-dextran by flow cytometry, after incubation with three viral proteins (5 μg/mL) for 3 h. *P<0.05.

Fig 5 S-RBD, N and NSP7 proteins of SARS-COV-2 stimulated the macropinocytosis in LPS-induced inflammatory cells

图 6 EIPA抑制正常细胞、LPS诱导的炎症细胞的巨胞饮水平，以及His标记的SARS-CoV-2原始株病毒蛋白的摄入Note：A. Co-localization of viral proteins and FITC-70 kDa-dextran in HEK-293T cells and LPS-induced HEK-293T cells by confocal microscopy, after preincubation with EIPA (×40). Scale bar=10 μm. B. With/without EIPA treatment, analysis of the mean fluorescence intensity of HEK-293T cells and LPS-induced HEK-293T cells internalized with FITC-70 kDa-dextran by high content screening, after incubation with three viral proteins (5 μg/mL) for 3 h. *P<0.05.

Fig 6 EIPA could inhibit the macropinocytosis level in normal cells and LPS-induced inflammatory cells, and the uptake of His-labeled SARS-CoV-2 proteins

图 7 Rab5 siRNA纳米药物载体抑制由SARS-COV-2原始株的病毒蛋白上调的细胞巨胞饮作用Note：A/B. Detection of mRNA expression (A) and protein expression (B) of Rab5 by qPCR and Western blotting, respectively. C. Analysis of the fluorescence level of HEK-293T cells internalized with FITC-70 kDa-dextran by flow cytometry, after incubation with three viral proteins and lipoprotein nano-drug carriers loaded with siRab5-1. *P<0.05.

Fig 7 Lipoprotein nano-drug carriers loaded with Rab5 siRNA could inhibit the macropinocytosis level up-regulated by SARS-COV-2 viral proteins

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巨胞饮途径介导的新冠原始株病毒蛋白入胞的细胞水平初步研究

Preliminary study on the cellular level of SARS-CoV-2 proteins mediated by macropinocytosis pathway

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