Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication.

The COVID-19 pandemic continues to threaten healthcare systems worldwide due to the limited access to vaccines, suboptimal treatment options, and the continuous emergence of new and more transmissible SARS-CoV-2 variants. Reverse-genetics studies of viral genes and mutations have proven highly valuable in advancing basic virus research, leading to the development of therapeutics. We developed a functional and highly versatile full-length SARS-CoV-2 infectious system by cloning the sequence of a COVID-19 associated virus isolate (DK-AHH1) into a bacterial artificial chromosome (BAC). Viruses recovered after RNA-transfection of in vitro transcripts into Vero E6 cells showed growth kinetics and remdesivir susceptibility similar to the DK-AHH1 virus isolate. Insertion of reporter genes, green fluorescent protein, and nanoluciferase into the ORF7 genomic region led to high levels of reporter activity, which facilitated high throughput treatment experiments. We found that putative coronavirus remdesivir resistance-associated substitutions F480L and V570L-and naturally found polymorphisms A97V, P323L, and N491S, all in nsp12-did not decrease SARS-CoV-2 susceptibility to remdesivir. A nanoluciferase reporter clone with deletion of spike (S), envelope (E), and membrane (M) proteins exhibited high levels of transient replication, was inhibited by remdesivir, and therefore could function as an efficient non-infectious subgenomic replicon system. The developed SARS-CoV-2 reverse-genetics systems, including recombinants to modify infectious viruses and non-infectious subgenomic replicons with autonomous genomic RNA replication, will permit high-throughput cell culture studies-providing fundamental understanding of basic biology of this coronavirus. We have proven the utility of the systems in rapidly introducing mutations in nsp12 and studying their effect on the efficacy of remdesivir, which is used worldwide for the treatment of COVID-19. Our system provides a platform to effectively test the antiviral activity of drugs and the phenotype of SARS-CoV-2 mutants.

SARS-CoV-2 replicon for high-throughput antiviral screening.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.

Generation of SARS-CoV-2 reporter replicon for high-throughput antiviral screening and testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a noninfectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlight the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

Engineering a Reliable and Convenient SARS-CoV-2 Replicon System for Analysis of Viral RNA Synthesis and Screening of Antiviral Inhibitors.

The etiologic agent of COVID-19 is highly contagious and has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. In this study, we reported a replicon system which consists of four plasmids expressing the required segments of SARS-CoV-2. Our study revealed that the features for viral RNA synthesis and responses to antivirus drugs of the replicon are similar to those of wild-type viruses. Further analysis indicated that ORF6 provided potent in trans stimulation of the viral replication. Some viral variations, such as 5'UTR-C241T and ORF8-(T28144C) L84S mutation, also exhibit their different impact upon viral replication. Besides, the screening of clinically used drugs identified that several tyrosine kinase inhibitors and DNA-Top II inhibitors potently inhibit the replicon, as well as authentic SARS-CoV-2 viruses. Collectively, this replicon system provides a biosafety-worry-free platform for studying SARS-CoV-2 virology, monitoring the functional impact of viral mutations, and developing viral inhibitors.IMPORTANCE COVID-19 has caused a severe global pandemic. Until now, there has been no simple and reliable system available in a lower-biosafety-grade laboratory for SARS-CoV-2 virologic research and inhibitor screening. We reported a replicon system which consists of four ordinary plasmids expressing the required segments of SARS-CoV-2. Using the replicon system, we developed three application scenarios: (i) to identify the effects of viral proteins on virus replication, (ii) to identify the effects of mutations on viral replication during viral epidemics, and (iii) to perform high-throughput screening of antiviral drugs. Collectively, this replicon system would be useful for virologists to study SARS-CoV-2 virology, for epidemiologists to monitor virus mutations, and for industry to develop antiviral drugs.

A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2.

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-α), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression to zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.