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

ABSTRACT

Objective·To investigate the effects of several key proteins of severe acute respiratory syndrome coronavirus 2 (SARSCoV-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. © 2022 Editorial Department of Journal of Shanghai Second Medical University. All rights reserved.