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.

Keywords: (+) ssRNA viruses; Key-words: replicon systems; direct-acting antivirals; drug discovery.

Figure 1

Schematic representation of a viral infection (a) compared to the transfection of cells with subgenomic RNA replicon systems (b). In (a), viral particles (1) are recognized by cell receptors and enter the cell via endocytosis (2). The acid environment of endosomes leads to the reorganization of the envelope glycoprotein (3), allowing the fusion of viral and endosomal membranes and the release of viral (+) ssRNA into the cytosol (4). Viral RNA hijacks the host cell’s translation machinery (5) to produce structural and nonstructural proteins (6). The nonstructural proteins assemble at the endoplasmic reticulum to form the replication complex together with host cell factors (7), allowing the viral RNA replication. Nascent viral RNA is englobed by viral structural proteins, forming immature viral particles (8). Immature viral particles travel through the Golgi apparatus, where pH and enzymatic modifications allow particle maturation and release. In (b) in vitro transcribed replicon RNA (1) is transfected into susceptible mammalian cells. In this case, electroporation is represented by the rays because it is the most widely used physical method able to transfect a large number of cells in a short time, as it is an easy and rapid transfection option [16]. (2) Replicon RNA harbors a reporter gene in place with structural protein genes. (3) Replicon RNA hijacks the host cell’s translation machinery to produce the reporter protein and nonstructural proteins. (4) Nonstructural proteins assemble at the endoplasmic reticulum to form the replication complex together with host cell factors, allowing the replicon RNA replication (5). (6) The reporter protein in the cytoplasm produces a fluorescent or luminescent signal that correlates with the level of accumulated replicon RNA, allowing the follow-up of the replicon replication efficiency.

Figure 2

Schematic representation of enveloped RNA viruses’ genome and subgenomic replicon systems. (a) Flaviviridae replicon based on the construct described by [8] for the Zika virus (ZIKV). C₃₈ and E₃₀ represent DNA sequences encoding the first 38 amino acids of C protein and the last 30 amino acids of E protein, respectively. A fragment containing the internal ribosomal entry site (IRES) and Neo gene was inserted downstream of the first 28 nucleotides of 3′UTR. Rluc—Renilla luciferase; HDVr—hepatitis delta virus ribozyme sequence. (b) Togaviridae replicon based on the construct described by [24] for the Chikungunya virus (CHIKV). A cassette encoding Pac fused to the enhanced green fluorescent protein (EGFP) via the 2A autoprotease of the foot-and-mouth disease virus (FMDV) was inserted under the control of the sg-promoter. In addition, the coding sequence of Rluc was inserted after the codon for amino acid 1823 of P1234 reading frame (after codon 490 of nsP3). (c) Coronaviridae replicon based on the construct described by [25] for Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). The nucleocapsid gene, N, was retained because the encoded protein has been shown to be required for viral RNA synthesis. Green fluorescent protein–blasticidin deaminase fusion (GFP–BlaR) gene was inserted between ORF 1 and N, not at the 5′ or 3′ end of the replicon, in order to minimize any possible deleterious effects in the synthesis of replicon RNA. L—leader sequence.

Figure 3

Schematic representation of non-enveloped RNA viruses’ genomes and subgenomic replicon systems. (a) Picornaviridae replicon based on the construct described by [83] for the Poliovirus (PV). The capsid coding P1 region is replaced by the Rluc gene. (b) Hepeviridae replicon based on the construct described by [84] for the Hepatitis E virus (HEV). The ORF2 capsid gene is disrupted with the Fluc gene (nucleotides 5148 to 5816). (c) Caliciviridae replicon based on the construct described by [85] for the Norwalk virus (NV). The Neo gene was engineered into the 5′-end region of the ORF2 so that the expressed product would contain the first 33 aa of the NV VP1 fused in frame with neomycin phosphotransferase.

Figure 4

Two-dimensional structures of approved antivirals against single-stranded positive RNA ((+) ssRNA) viruses.