Mechanisms Of Cell Cycle Arresting Compounds Promoting SARS-CoV-2 Cell Entry Through Upregulation Of ACE2

As the receptor for SARS-CoV-2 entry,expression level of ACE2 is closely related to SARS-CoV-2 infection.Multiple studies have shown that ACE2 could regulate cell proliferation.However,little is known about how cell proliferation state affects the expression level of ACE2.CDK4/6 inhibitor palbociclib was reported to be one of the topscored repurposed drugs to treat COVID-19.In this study,we aimed to investigate the effect of palbociclib and other cell cycle arresting compounds on the regulation of ACE2 expression and subsequent viral infection,providing a basis for the treatment of COVID19.Four methods were used to achieve cell cycle arrest in multiple cell lines.At the transcriptional level,we used RT-qPCR to detect changes in ACE2 mRNA levels,and further explored the transcriptional regulation of STAT3 and c-Jun transcription factors predicted by bioinformatics analysis tools on ACE2 using RT-qPCR,western blot,and dual luciferase reporter gene assays.At the protein level,we conducted an in-depth study of the protein degradation pathway of ACE2 using western blot,immunofluorescence,flow cytometry,and bioinformatics analysis.Finally,we explored the effects of cell cycle arresting drugs on SARS-CoV-2 infection using SARS-CoV-2 pseudoviral infection model in both Huh-7 and Vero E6 cells.We demonstrated that palbociclib and other methods could arrest cells in G0/G1 phase and up-regulate ACE2 mRNA and protein levels without altering its subcellular localization.At the transcriptional level,palbociclib and other cell cycle arresting compounds could upregulate expression levels of STAT3 and c-Jun,followed by activation of ACE2 promoter and elevation of ACE2 mRNA levels.At the protein level,palbociclib inhibited ubiquitin-proteasome and lysosomal degradation of ACE2 through down-regulating SKP2.In addition,increased ACE2 expression induced by cell cycle arrest facilitated SARS-CoV-2 pseudoviral infection.In conclusion,we demonstrated that ACE2 expression was down-regulated in proliferating cells.Cell cycle inhibitors could increase ACE2 expression through promoting mRNA levels and inhibiting protein degradation of ACE2 and facilitate SARS-CoV-2 infection,which might not work as effective therapeutic agents to treat SARS-CoV-2 infection.