Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses

Abstract

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.

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

“…Over the past decades, due in large part to the advances in reverse genetics, replicon systems have been established for a number of RNA viruses, such as hepatitis C virus, SARS-CoV-1, Middle East respiratory syndrome coronavirus, dengue virus, West Nile virus, Zika virus, poliovirus enterovirus 71, hepatitis E virus, chikungunya virus, and many others ( 26 ). For highly virulent viruses, the replicon assay system provides a safe and high-throughput method to screen and evaluate compounds.…”

Construction and characterization of two SARS‐CoV‐2 minigenome replicon systems

“…In addition, reporter genes can be engineered into replicons for high sensitivity and ease of detection, especially for high throughput antiviral screening purposes. 54 Compared to in vitro enzymatic assay‐based antiviral screening platforms, a replicon system is advantageous in the assessment of compound cytotoxicity and combination treatments, selection of drug resistant mutants, and discovery and evaluation of host targeting antiviral agents.…”

“…The viral subgenomic replicons comprising nonstructural genes essential to viral RNA replication but without structural genes are convenient tools when in vitro infection systems are unavailable or downgrade of biosafety levels is needed. In addition, reporter genes can be engineered into replicons for high sensitivity and ease of detection, especially for high throughput antiviral screening purposes 54 . Compared to in vitro enzymatic assay‐based antiviral screening platforms, a replicon system is advantageous in the assessment of compound cytotoxicity and combination treatments, selection of drug resistant mutants, and discovery and evaluation of host targeting antiviral agents.…”

“…In addition, reporter genes can be engineered into replicons for high sensitivity and ease of detection, especially for high throughput antiviral screening purposes. 54 Compared to in vitro enzymatic assay-based antiviral screening platforms, a replicon system is advantageous in the assessment of compound cytotoxicity and Reverse genetics approaches were used to generate SARS-CoV-2 infectious clone and replicon systems immediately after the whole viral genome sequence became publicly available. 24,40 Due to the large size of SARS-CoV-2 genome, the viral cDNA genome was designed as split fragments of ∼5 kb each with suitable type IIS restriction sites located at the junctions, then the chemically synthesized fragments were subjected to restriction enzyme digestion and ligated into cDNA in vitro.…”

Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition

“…Over the past decades, due in large part to the advances in reverse genetics, replicon systems have been established for a number of RNA viruses, such as hepatitis C virus, SARS-CoV-1, middle east respiratory syndrome coronavirus (MERS-CoV), dengue virus, West Nile virus, Zika virus, poliovirus enterovirus 71, hepatitis E virus, chikungunya virus and many others ( 24 ). For highly virulent viruses, the replicon assay system provides a safe and high throughput method to screen and evaluate compounds.…”