Aixin Li1, Kaitao Zhao1, Bei Zhang1, Rong Hua1, Yujie Fang2,3, Wuhui Jiang1, Jing Zhang4, Lixia Hui1, Yingcheng Zheng1, Yan Li5,6, Chengliang Zhu7, Pei-Hui Wang4, Ke Peng2, Yuchen Xia1. 1. State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, School of Basic Medical Sciences, Wuhan University, Wuhan, China. 2. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei 430071, China. 3. University of Chinese Academy of Sciences, Beijing, China. 4. Key Laboratory for Experimental Teratology of Ministry of Education and Advanced Medical Research Institute, Cheeloo College of Medicine, Shandong University, 250012, Jinan, China. 5. Department of Pathogen Biology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 6. Tongji-Rongcheng Center for Biomedicine, Huazhong University of Science and Technology, Wuhan, China. 7. Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, China.
Abstract
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 non-structural protein 12 (NSP12) was able to suppress interferon-β (IFN-β) activation in IFN-β promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-β promoter mediated luciferase activity was reduced during co-expression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-β production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-β promoter luciferase assays. In conclusion, unlike previously reported, our study showed SARS-CoV-2 NSP12 protein is not an IFN-β antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. Importance Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling, and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-β activation. However, most of these results were generated from IFN-β promoter luciferase reporter assay, and have not been validated functionally. In our study, we found that although NSP12 could suppress IFN-β promoter luciferase activity, it showed no inhibitory effect on IFN-β production or it downstream signaling. Further study revealed that contradictory results could generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2 NSP12 could not suppress IFN-β signaling. On the other hand, our study suggests that cautions need to be taken with the interpretation of SARS-CoV-2 related luciferase assays.
The n class="Disease">coronavirus disease 2019 (n class="Disease">COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is bringing an unprecedented health crisis to the world. To date, our understanding of the interaction between SARS-CoV-2 and host innate immunity is still limited. Previous studies reported that SARS-CoV-2 non-structural protein 12 (NSP12) was able to suppress interferon-β (IFN-β) activation in IFN-β promoter luciferase reporter assays, which provided insights into the pathogenesis of COVID-19. In this study, we demonstrated that IFN-β promoter mediated luciferase activity was reduced during co-expression of NSP12. However, we could show NSP12 did not affect IRF3 or NF-κB activation. Moreover, IFN-β production induced by Sendai virus (SeV) infection or other stimulus was not affected by NSP12 at mRNA or protein level. Additionally, type I IFN signaling pathway was not affected by NSP12, as demonstrated by the expression of interferon stimulated genes (ISGs). Further experiments revealed that different experiment systems, including protein tags and plasmid backbones, could affect the readouts of IFN-β promoter luciferase assays. In conclusion, unlike previously reported, our study showed SARS-CoV-2NSP12 protein is not an IFN-β antagonist. It also rings the alarm on the general usage of luciferase reporter assays in studying SARS-CoV-2. Importance Previous studies investigated the interaction between SARS-CoV-2 viral proteins and interferon signaling, and proposed that several SARS-CoV-2 viral proteins, including NSP12, could suppress IFN-β activation. However, most of these results were generated from IFN-β promoter luciferase reporter assay, and have not been validated functionally. In our study, we found that although NSP12 could suppress IFN-β promoter luciferase activity, it showed no inhibitory effect on IFN-β production or it downstream signaling. Further study revealed that contradictory results could generated from different experiment systems. On one hand, we demonstrated that SARS-CoV-2NSP12 could not suppress IFN-β signaling. On the other hand, our study suggests that cautions need to be taken with the interpretation of SARS-CoV-2 related luciferase assays.