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ADAR1p110 promotes Enterovirus D68 replication through its deaminase domain and inhibition of PKR pathway.
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.
Afiliação
  • Zhang K; Pathogen Biology and Immunology Laboratory and Laboratory of Tissue and Cell Biology, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing, China.
  • Wang S; Department of the First Clinical Medicine, Chongqing Medical University, Chongqing, China.
  • Chen T; Department of the First Clinical Medicine, Chongqing Medical University, Chongqing, China.
  • Tu Z; Pathogen Biology and Immunology Laboratory and Laboratory of Tissue and Cell Biology, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing, China.
  • Huang X; Department of Pathogen Biology, Basic Medical School, Chongqing Medical University, Chongqing, China.
  • Zang G; Department of the First Clinical Medicine, Chongqing Medical University, Chongqing, China.
  • Wu C; Pathogen Biology and Immunology Laboratory and Laboratory of Tissue and Cell Biology, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing, China.
  • Fan X; Chongqing Better Biotechnology LLC, Chongqing, China.
  • Liu J; Department of the First Clinical Medicine, Chongqing Medical University, Chongqing, China.
  • Tian Y; Pathogen Biology and Immunology Laboratory and Laboratory of Tissue and Cell Biology, Experimental Teaching and Management Center, Chongqing Medical University, Chongqing, China.
  • Cheng Y; Quality Management Section, Chongqing Blood Center, Chongqing, China.
  • Lu N; Monitoring On Terrestrial Wildlife-Borne Infectious Diseases, Jinggangshan National Nature Reserve of Jiangxi Province, Ji'an, Jiangxi, China.
  • Zhang G; Department of Pathogen Biology, Basic Medical School, Chongqing Medical University, Chongqing, China. ficus@cqmu.edu.cn.
Virol J ; 19(1): 222, 2022 12 22.
Article em En | MEDLINE | ID: mdl-36550502
ABSTRACT

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 2α (p-eIF2α) 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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Enterovirus Humano D / Infecções por Enterovirus Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Virol J Assunto da revista: VIROLOGIA Ano de publicação: 2022 Tipo de documento: Article País de afiliação: China

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Enterovirus Humano D / Infecções por Enterovirus Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Virol J Assunto da revista: VIROLOGIA Ano de publicação: 2022 Tipo de documento: Article País de afiliação: China