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

doi:10.3969/j.issn.1674-8115.2022.08.003

Journal of Shanghai Jiao Tong University (Medical Science) ›› 2022, Vol. 42 ›› Issue (8): 987-996.doi: 10.3969/j.issn.1674-8115.2022.08.003

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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)

Abstract

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

CLC Number:

R373.1

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.

Figures/Tables 8

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

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

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

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

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

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

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

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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References 22

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

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