key: cord-1045783-wj5twfmm
authors: Xiu, Huiqing; Gong, Jiali; Huang, Tiancha; Peng, Yanmei; Bai, Songjie; Xiong, Guirun; Zhang, Shufang; Huang, Huaqiong; Cai, Zhijian; Zhang, Gensheng
title: Fludarabine inhibits type I interferon-induced expression of the SARS-CoV-2 receptor angiotensin-converting enzyme 2
date: 2021-05-31
journal: Cell Mol Immunol
DOI: 10.1038/s41423-021-00698-5
sha: af41c5560cdc24ae091cb14522ab53d7ba938c6c
doc_id: 1045783
cord_uid: wj5twfmm

nan

silencing or overexpression abolished or promoted, respectively, the IFN-α-induced increases in the levels of ACE2 gene and protein expression in both HBE cells (Fig. 1f-i ; respectively) and HEK293 cells (Fig. S2a , b, c, d; respectively). Next, we separately cloned four sequential Ace2 promoters (each with 450 bp) into a luciferase reporter plasmid, and the resulting constructs were termed pGL3-ACE2-1-Luc, pGL3-ACE2-2-Luc, pGL3-ACE2-3-Luc, and pGL3-ACE2-4-Luc. Overexpression of STAT1 significantly enhanced the luciferase activity of pGL3-ACE2-3-Luc (containing the Ace2 promoter region including nucleotides −1250 to −833) ( Fig. 1j ) but not the other three luciferase reporter plasmids (Fig. S2e ). Further chromatin immunoprecipitation experiments showed that the Ace2 promoter region containing nucleotides −1232 to −1032 was enriched by precipitation with an anti-STAT1 antibody and enhanced by IFN-α stimulation (Fig. 1k) ; this region was within the scope of the in silico bioinformatic analysis (−1500 to −500 bp upstream of the transcription start site). 9 These results indicate that IFN-α induces direct binding of STAT1 to the Ace2 promoter and subsequently enhances transcription of the Ace2 gene.

Fludarabine is an inhibitor of STAT1 and a common chemotherapeutic drug used to treat chronic B lymphocytic leukemia in the clinic 10 ; we presumed that it could reduce ACE2 expression by inhibiting STAT1. At a low concentration (<1 μM), fludarabine did not influence the proliferation of these cells within 24 h (Fig. S3a) , whereas it obviously decreased the IFN-α-induced upregulation of ACE2 gene and protein expression in HBE cells (Fig. 1l , m) and in A549, HEK293, and HCC-LM3 cells (Fig. S3b, c) , accompanied by a notable reduction in both p-STAT1 and STAT1 protein levels. Furthermore, the levels of both intracellular and membrane ACE2 protein were reduced by fludarabine treatment in IFN-αstimulated HBE cells (Fig. 1n , o) and in A549 and HCC-LM3 cells (Fig. S3d, e) . In contrast, these effects were abolished in HBE cells after STAT1 knockdown (Fig. 1p) . These results demonstrate that fludarabine reduces IFN-α-induced ACE2 expression in a STAT1dependent manner.

In conclusion, we provide direct in vitro evidence that type I interferon IFN-α promotes STAT1 expression and phosphorylation and that phosphorylated STAT1 upregulates the expression of the Ace2 gene and ACE2 protein by binding to the promoter region (positions −1232 to −1032) of Ace2, an effect that can be Fig. 1 Fludarabine inhibits IFN-α-induced ACE2 expression via STAT1. a Immunoblot analysis of ACE2 in A549 and HBE cells treated with IFN-α for 6, 12, and 24 h. b, c After treatment with IFN-α for 12 h, ACE2 protein expression in A549 and HBE cells was detected by immunofluorescence (b) and flow cytometry (c). Scale bars = 10 μm. d Real-time PCR analysis of Ace2 gene expression in A549 and HBE cells treated with IFN-α. e HBE cells were treated with CHX (50 μM) and IFN-α for the indicated times and harvested for immunoblot analysis. f, g HBE cells were transfected with NC or Stat1 siRNA for 48 h and were then treated with IFN-α for 12 h. Cells were harvested for immunoblot (f) and real-time PCR (g) analyses (n = 3). h, i HBE cells were transfected with mock or Stat1 overexpression plasmids for 24 h and were then treated with IFN-α for another 12 h. Cells were harvested for immunoblot (h) and real-time PCR (i) analyses (n = 3). j Dual luciferase assay to evaluate the potential regulation of Ace2 promoter activity by STAT1 in 293T cells after cotransfection with pGL3-Ace2-3-luci and Stat1 expression plasmids and the Renilla luciferase reporter plasmid or empty vector for 24 h (n = 5). k ChIP analysis of the binding of STAT1 to the Ace2 promoter at positions −1232 to −1032 in HEK293 cells under normal or IFN-α stimulation conditions (n = 3). l-o HBE cells were treated with or without fludarabine (1 μM) for 12 h and were then treated with or without IFN-α for another 12 h. Cells were harvested for immunoblot (l), real-time PCR (m), immunofluorescence (n) and flow cytometric (o) analyses. Scale bar = 10 μm. p HBE cells were transfected with NC or Stat1 siRNA for 48 h and were then treated with fludarabine (1 μM) for 12 h and with or without IFN-α for another 12 h. Cells were harvested for immunoblot analysis. The data shown are the mean ± SD values and are representative of three independent experiments. Student's t test was used for statistical analysis (n = 3). The concentration of IFN-α used in all in vitro experiments was 100 ng/ml. *P < 0.05; **P < 0.01; ***P < 0.001 

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Increasing host cellular receptor-angiotensin-converting enzyme 2 expression by coronavirus may facilitate 2019-nCoV (or SARS-CoV-2) infection

SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues

A road map for those who don't know JAK-STAT

Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling

Methyltransferase SETD2-mediated methylation of STAT1 is critical for interferon antiviral activity

Bioinformatic characterization of angiotensin-converting enzyme 2, the entry receptor for SARS-CoV-2

A virus-induced conformational switch of STAT1-STAT2 dimers boosts antiviral defenses

Competing interests: The authors declare no competing interests.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/.