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

Abstract

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 of 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.

Interferon Control of Human Coronavirus Infection and Viral Evasion: Mechanistic Insights and Implications for Antiviral Drug and Vaccine Development

“… [167] , [229] ZAP Restricts SARS-CoV-2 replication. Binds CpG dinucleotides in viral RNA to mark it for degradation [142] , [143] , [144] OAS1-RNase L Restricts SARS-CoV-2 replication. Synthesize 2’-5’oligo (A) that actives RNase L to degrade viral and cellular RNA.…”

“…Recent studies indicated that ZAP restricts SARS-CoV-2 by targeting CpG dinucleotides in viral RNA sequences [142] , [143] , [144] . Interestingly, SARS-CoV-2 and two closely-related animal CoVs isolated from horseshoe bats, RaTG13 and RmYN02, exhibited remarkable CpG suppression in comparison to other human and bat CoVs [142] .…”

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

“…Molnupiravir reduces hospitalization or death by 30% (21,22). Viral proteases have also been excellent targets for the development of antiviral drugs (23,24).…”

Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening

“…CoV-2 replicon RNA under the control of the CMV promoter (Nguyen et al, 2021), while others have made transient replicon systems using in vitro-transcribed SARS-CoV-2 replicon RNA (He et al, 2021;Kotaki et al, 2021;Zhang et al, 2021). In each replicon system, the expression of luciferase or a fluorescent reporter gene of replicon RNA is induced in a replication-dependent manner.…”

“…Since experimental infection with SARS-CoV-2 must be restricted under the condition of biosafety level 3 (BSL3), BSL2-compatible SARS-CoV-2 replicon systems have been developed instead of in vitro infection models for basic research and antiviral screening (He et al, 2021;Kotaki et al, 2021;Nguyen et al, 2021;Zhang et al, 2021).…”