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Construction and characterization of two SARS-CoV-2 minigenome replicon systems.

ABSTRACT: The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology and antiviral discovery, a noninfectious 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 complementary DNA template to generate a replicon messenger RNA (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 suggest 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.

SUBMITTER: Zhang H

PROVIDER: S-EPMC9088700 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Construction and characterization of two SARS-CoV-2 minigenome replicon systems.

Zhang Hu H Fischer Douglas K DK Shuda Masahiro M Moore Patrick S PS Gao Shou-Jiang SJ Ambrose Zandrea Z Guo Haitao H

Journal of medical virology 20220219 6

The ongoing COVID-19 pandemic severely impacts global public health and economies. To facilitate research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virology and antiviral discovery, a noninfectious 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 complementary DNA template to generate a replicon messeng ...[more]

PMID: 35137972

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Project description:The emerging coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide, resulting in global public health emergencies and economic crises. In the present study, a noninfectious and biosafety level 2 (BSL2)-compatible SARS-CoV-2 replicon expressing a nano luciferase (nLuc) reporter was constructed in a bacterial artificial chromosomal (BAC) vector by reverse genetics. The nLuc reporter is highly sensitive, easily quantifiable, and high throughput adaptable. Upon transfecting the SARS-CoV-2 replicon BAC plasmid DNA into Vero E6 cells, we could detect high levels of nLuc reporter activity and viral RNA transcript, suggesting the replication of the replicon. The replicon replication was further demonstrated by the findings that deleting nonstructural protein 15 or mutating its catalytic sites significantly reduced replicon replication, whereas providing the nucleocapsid protein in trans enhanced replicon replication in a dose-dependent manner. Finally, we showed that remdesivir, a U.S. Food and Drug Administration-approved antiviral drug, significantly inhibited the replication of the replicon, providing proof of principle for the application of our replicon as a useful tool for developing antivirals. Taken together, this study established a sensitive and BSL2-compatible reporter system in a single BAC plasmid for investigating the functions of SARS-CoV-2 proteins in viral replication and evaluating antiviral compounds. This should contribute to the global effort to combat this deadly viral pathogen. IMPORTANCE The COVID-19 pandemic caused by SARS-CoV-2 is having a catastrophic impact on human lives. Combatting the pandemic requires effective vaccines and antiviral drugs. In the present study, we developed a SARS-CoV-2 replicon system with a sensitive and easily quantifiable reporter. Unlike studies involving infectious SARS-CoV-2 virus that must be performed in a biosafety level 3 (BSL3) facility, the replicon is noninfectious and thus can be safely used in BSL2 laboratories. The replicon will provide a valuable tool for testing antiviral drugs and studying SARS-CoV-2 biology.

Project description:Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the EBOV minigenome demonstrated that the stable EBOV replicon had much-more-active minigenome replication than previously developed transient-transfection-based EBOV minigenome systems, which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients.IMPORTANCE The scope and severity of the recent Ebola outbreak in Western Africa justified a more comprehensive investigation of the causative risk group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows experimentation under biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The replicon system had more-active genome replication than previously developed transient-transfection-based EBOV minigenome systems, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing, and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.

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Construction and characterization of two SARS-CoV-2 minigenome replicon systems.

Publications

Construction and characterization of two SARS-CoV-2 minigenome replicon systems.

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