ADAR1p110 promotes Enterovirus D68 replication through its deaminase domain and inhibition of PKR pathway

作者全名："Zhang, Kehan; Wang, Siyuan; Chen, Tingting; Tu, Zeng; Huang, Xia; Zang, Guangchao; Wu, Chun; Fan, Xinyue; Liu, Jia; Tian, Yunbo; Cheng, Yong; Lu, Nan; Zhang, Guangyuan"

作者地址："[Zhang, Kehan; Chen, Tingting; Zang, Guangchao; Liu, Jia; Zhang, Guangyuan] Chongqing Med Univ, Expt Teaching & Management Ctr, Pathogen Biol & Immunol Lab, Chongqing, Peoples R China; [Zhang, Kehan; Chen, Tingting; Zang, Guangchao; Liu, Jia; Zhang, Guangyuan] Chongqing Med Univ, Expt Teaching & Management Ctr, Lab Tissue & Cell Biol, Chongqing, Peoples R China; [Tu, Zeng; Lu, Nan] Chongqing Med Univ, Basic Med Sch, Dept Pathogen Biol, Chongqing, Peoples R China; [Zhang, Kehan; Wang, Siyuan; Huang, Xia; Fan, Xinyue] Chongqing Med Univ, Dept Clin Med 1, Chongqing, Peoples R China; [Tian, Yunbo] Chongqing Blood Ctr, Qual Management Sect, Chongqing, Peoples R China; [Wu, Chun] Chongqing Better Biotechnol LLC, Chongqing, Peoples R China; [Cheng, Yong] Jinggangshan Natl Nat Reserve Jiangxi Prov, Monitoring Terr Wildlife Borne Infect Dis, Jian, Jiangxi, Peoples R China"

通信作者："Zhang, GY (通讯作者)，Chongqing Med Univ, Expt Teaching & Management Ctr, Pathogen Biol & Immunol Lab, Chongqing, Peoples R China.; Zhang, GY (通讯作者)，Chongqing Med Univ, Expt Teaching & Management Ctr, Lab Tissue & Cell Biol, Chongqing, Peoples R China.; Lu, N (通讯作者)，Chongqing Med Univ, Basic Med Sch, Dept Pathogen Biol, Chongqing, Peoples R China."

来源：VIROLOGY JOURNAL

ESI学科分类：MICROBIOLOGY

WOS号：WOS:000903248200001

JCR分区：Q2

影响因子：4.8

年份：2022

卷号：19

期号：1

开始页：　

结束页：　

文献类型：Article

关键词：EV-D68; ADAR1p110; Deaminase domain; dsRBDs; PKR

摘要："Background: Severe respiratory and neurological diseases caused by human enterovirus D68 (EV-D68) pose a serious threat to public health, and there are currently no effective drugs and vaccines. Adenosine deaminase acting on RNA1 (ADAR1) has diverse biological functions in various viral infections, but its role in EV-D68 infections remains undetermined.Methods: Rhabdomyosarcoma (RD) and human embryonic kidney 293 T (293 T) cells, and HeLa cells were used to evaluate the expression level of ADAR1 upon EV-D68 (Fermon strain) and human parainfluenza virus type 3 (HPIV3; NIH47885) infection, respectively. Knockdown through silencing RNA (siRNA) and overexpression of either ADAR1p110 or ADAR1p150 in cells were used to determine the function of the two proteins after viral infection. ADAR1p110 double-stranded RNA binding domains (dsRBDs) deletion mutation was generated using a seamless clone kit. The expression of ADAR1, EV-D68 VP1, and HPIV3 hemagglutinin-neuraminidase (HN) proteins was identified using western blotting. The median tissue culture infectious dose (TCID50) was applied to detect viral titers. The transcription level of EV-D68 mRNA was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and the viral 5 '-untranslated region (5 '-UTR)-mediated translation was analyzed using a dual luciferase reporter system.Conclusion: We found that the transcription and expression of ADAR1 was inhibited upon EV-D68 infection. RNA interference of endogenous ADAR1 decreased VP1 protein expression and viral titers, while overexpression of ADAR1p110, but not ADAR1p150, facilitated viral replication. Immunofluorescence assays showed that ADAR1p110 migrated from the nucleus to the cytoplasm after EV-D68 infection. Further, ADAR1p110 lost its pro-viral ability after mutations of the active sites in the deaminase domain, and 5 '-UTR sequencing of the viral genome revealed that ADAR1p110 likely plays a role in EV-D68 RNA editing. In addition, after ADAR1 knockdown, the levels of both phosphorylated double-stranded RNA dependent protein kinase (p-PKR) and phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2 alpha) increased. Attenuated translation activity of the viral genome 5 '-UTR was also observed in the dual-luciferase reporter assay. Lastly, the deletion of ADAR1p110 dsRBDs increased the level of p-PKR, which correlated with a decreased VP1 expression, indicating that the promotion of EV-D68 replication by ADAR1p110 is also related to the inhibition of PKR activation by its dsRBDs. Our study illustrates that ADAR1p110 is a novel pro-viral factor of EV-D68 replication and provides a theoretical basis for EV-D68 antiviral research."

基金机构："Chongqing Yuzhong District Science and Technology Commission; Undergraduate Innovation Experi-ment Project [20190123]; Tutorial System of Excellent Medical Undergraduateof Chongqing Medical University [SRIEP201958, SRIEP202007]; Chongqing Municipal Education Commission Foundation [LTMC-MTS202114]; Natural Science Foundation of Chongqing [KJQN202000403]; Chongqing Municipal Science and Health Joint Research Project [cstc2021jcyj-msxmX0095]; [2019MSXM048]"

基金资助正文："This work was supported by the Chongqing Yuzhong District Science and Technology Commission (20190123), the Undergraduate Innovation Experi-ment Project (SRIEP201958 and SRIEP202007) and the Tutorial System of Excellent Medical Undergraduateof Chongqing Medical University (LTMC-MTS202114), the Chongqing Municipal Education Commission Foundation (KJQN202000403), Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0095) and Chongqing Municipal Science and Health Joint Research Project (2019MSXM048)"