Viral replicons as valuable tools for drug discovery

Abstract

RNA viruses can cause severe diseases such as dengue, Lassa, chikungunya and Ebola. Many of these viruses can only be propagated under high containment levels, necessitating the development of low containment surrogate systems such as subgenomic replicons and minigenome systems. Replicons are self-amplifying recombinant RNA molecules expressing proteins sufficient for their own replication but which do not produce infectious virions. Replicons can persist in cells and are passed on during cell division, enabling quick, efficient and high-throughput testing of drug candidates that act on viral transcription, translation and replication. This review will explore the history and potential for drug discovery of hepatitis C virus, dengue virus, respiratory syncytial virus, Ebola virus and norovirus replicon and minigenome systems.

Figure 1

Graphic representation of viral genomes and their derived replicons and minigenomes. Each region coding for a protein is shown as a box in red if the region codes for a structural protein, in yellow if it codes for a nonstructural (NS) protein or in purple if it codes for a nonviral protein. Internal ribosome entry sites (IRES) and the foot-and-mouth disease virus 2A autoprotease sequences (FMDV2A) are marked by lines or arrows, respectively. For minigenomes (d,f) plasmid-driven expression of viral genes is shown by circularized lines. (a) Hepatitis C virus (HCV) genome (ai) and its derived replicon (aii). The HCV replicon was established by replacement of C, E1, E2 and NS2 by the neomycin phosphotransferase (neo). Translation of NS3–NS5B was driven by an IRES sequence. (b) Dengue virus (DENV) genome (bi) and its derived replicons (bii and iii). Replicons for dengue virus have been produced by replacement of the C-terminal part of C, full length prM and the N-terminal part of E with either green fluorescent protein (GFP), which is cleaved by from NS1 by the FMDV2A sequence (bii), or with a polyprotein cleaved by the FMDV2A sequence producing puromycin-N-acetyl transferase (pac) and enhanced GFP (EGFP). Translation of NS1–NS5 is driven by an IRES sequence (biii). (c)Togaviridae genome (ci) and derived replicons for Chikungunya virus (CHIKV) (cii and ciii) and Sindbis virus (SINV) (civ). Replicons have been established by replacement of the structural polyprotein (C-E3-E2-6K-E1) with the polyprotein cleaved by FMDV2A producing pac and EGFP (cii) or an additional insertion of Renilla luciferase (RLuc) within nonstructural protein (nsp) 3 (ciii). The SINV replicon was established by replacement of the structural polyprotein with pac. (d) Respiratory syncytial virus genome (di) and its derived minigenome system (dii) or replicon (diii). The minigenome system was established by tagging the coding sequence of chloramphenicol acetyl-transferase (cat) with the 5′ leader and the 3′ trailer sequences of the viral genome and plasmid-driven co-expression of the viral polymerase (L), nucleoprotein (N), phosphoprotein (P) and matrix protein 2 (M2). The replicon (diii) was established by deletion of the coding regions of the small hydrophobic protein (SH) and the two glycoproteins G and F, and insertion of GFP at the 5′ end of NS1. (e) Human norovirus (NV) genome (ei) and its derived replicon (eii). The replicon was established by replacement of the majority of the viral protein (VP)1 coding region with neo. (f) Ebola virus (EBOV) genome shown in (+) sense (fi) and its derived minigenome system (fii). The minigenome was established by tagging GFP with the (+) sense 5′ leader and 3′ trailer sequences of the viral genome and plasmid-driven co-expression of the viral polymerase (L), VP30, VP35 and the nucleoprotein (NP).