tetano
Editor, Senior Moderator
J Med Virol
. 2022 Feb 9.
doi: 10.1002/jmv.27650. Online ahead of print.
Construction and characterization of two SARS-CoV-2 minigenome replicon systems
Hu Zhang[SUP] 1 2 [/SUP], Douglas K Fischer[SUP] 1 2 [/SUP], Masahiro Shuda[SUP] 1 2 [/SUP], Patrick S Moore[SUP] 1 2 [/SUP], Shou-Jiang Gao[SUP] 1 2 [/SUP], Zandrea Ambrose[SUP] 1 2 [/SUP], Haitao Guo[SUP] 1 2 [/SUP]
Affiliations
Abstract
The ongoing COVID-19 pandemic severely impacts global public health and economies. In order to facilitate research on SARS-CoV-2 virology and antiviral discovery, a non-infectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS-CoV-2 minigenome replicon systems. First, we constructed the IVT-CoV2-Rep cDNA template to generate a replicon mRNA with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT-CoV2-Rep in a variety of cell lines, which could be completely abolished by known SARS-CoV-2 replication inhibitors. Our data also suggests that the transient phenotype of IVT-CoV2-Rep is not due to host innate antiviral responses. In addition, we have developed a DNA-launched replicon BAC-CoV2-Rep, which supports the in-cell transcription of a replicon mRNA as initial replication template. The BAC-CoV2-Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC-CoV2-Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS-CoV-2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization. This article is protected by copyright. All rights reserved.
Keywords: Antiviral agents; Cellular Effect; Disease control; Immune responses; Innate immunity; SARS coronavirus; Virus classification; mRNA/Splicing.
. 2022 Feb 9.
doi: 10.1002/jmv.27650. Online ahead of print.
Construction and characterization of two SARS-CoV-2 minigenome replicon systems
Hu Zhang[SUP] 1 2 [/SUP], Douglas K Fischer[SUP] 1 2 [/SUP], Masahiro Shuda[SUP] 1 2 [/SUP], Patrick S Moore[SUP] 1 2 [/SUP], Shou-Jiang Gao[SUP] 1 2 [/SUP], Zandrea Ambrose[SUP] 1 2 [/SUP], Haitao Guo[SUP] 1 2 [/SUP]
Affiliations
- PMID: 35137972
- DOI: 10.1002/jmv.27650
Abstract
The ongoing COVID-19 pandemic severely impacts global public health and economies. In order to facilitate research on SARS-CoV-2 virology and antiviral discovery, a non-infectious viral replicon system operating under biosafety level 2 containment is warranted. We report herein the construction and characterization of two SARS-CoV-2 minigenome replicon systems. First, we constructed the IVT-CoV2-Rep cDNA template to generate a replicon mRNA with nanoluciferase (NLuc) reporter via in vitro transcription (IVT). The replicon mRNA transfection assay demonstrated a rapid and transient replication of IVT-CoV2-Rep in a variety of cell lines, which could be completely abolished by known SARS-CoV-2 replication inhibitors. Our data also suggests that the transient phenotype of IVT-CoV2-Rep is not due to host innate antiviral responses. In addition, we have developed a DNA-launched replicon BAC-CoV2-Rep, which supports the in-cell transcription of a replicon mRNA as initial replication template. The BAC-CoV2-Rep transient transfection system exhibited a much stronger and longer replicon signal compared to the IVT-CoV2-Rep version. We also found that a portion of the NLuc reporter signal was derived from the spliced BAC-CoV2-Rep mRNA and was resistant to antiviral treatment, especially during the early phase after transfection. In summary, the established SARS-CoV-2 transient replicon systems are suitable for basic and antiviral research, and hold promise for stable replicon cell line development with further optimization. This article is protected by copyright. All rights reserved.
Keywords: Antiviral agents; Cellular Effect; Disease control; Immune responses; Innate immunity; SARS coronavirus; Virus classification; mRNA/Splicing.