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.
Highlights
Single-stranded positive RNA ((+) ssRNA) viruses include a group of human pathogens with significant socioeconomic impacts, such as Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Hepatitis C virus (HCV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory SyndromeCoronavirus 2 (SARS-CoV-2), Poliovirus (PV), Enterovirus 71 (EV-71), Hepatitis E virus (HEV), and many others
The Rluc sequence was fused with Chikungunya virus (CHIKV) nsP3, and enhanced green fluorescent protein (EGFP) was produced as a fusion protein with puromycin N-acetyltransferase (Pac) under the control of sg-promoter [24]
Several replicon-based systems have been developed for the discovery of direct-acting antivirals (DAA) targeting (+) ssRNA viruses (Figure 4)
Summary
Single-stranded positive RNA ((+) ssRNA) viruses include a group of human pathogens with significant socioeconomic impacts, such as Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), Hepatitis C virus (HCV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome. This loss-of-signal end point can produce false-positive hits, resulting from molecules that interfere with the activity of the reporter protein and those that adversely affect cell health For this reason, a cytotoxicity assay, performed in parallel with the primary screen, assists in the discovery of specific viral inhibitors [15]. The reporter protein in the cytoplasm produces a fluorescent or cell factors, allowing the replicon RNA replication (5). RNA produces single-round (SRIPs), which are infectious, but progeny virusesbut cannot propagate from the infected cells, as the infectious particles (SRIPs), which are infectious, progeny viruses cannot propagate from the packaged genome lacks structural protein genes [17]. VRPs allow screening of antiviral compounds infected cells, as the packaged genome lacks structural protein genes [17]. We compiled the recent progress made in the use of replicon-based assays for the development of antivirals against (+) ssRNA viruses, highlighting some of the most promising inhibitors identified by the method to date
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