Construction of Self-Replicating Subgenomic Dengue Virus 4 (DENV4) Replicon

Abstract

Dengue virus serotypes 1–4 are members of mosquito-borne flavivirus genus of Flaviviridae family that encode one long open reading frame (ORF) that is translated to a polyprotein. Both host and virally encoded proteases function in the processing of the polyprotein by co-translational and posttranslational mechanisms to yield 10 mature proteins prior to viral RNA replication. To study cis- and trans-acting factors involved in viral RNA replication, many groups [1–8] have constructed cDNAs encoding West Nile virus (WNV), DENV, or yellow fever virus reporter replicon RNAs. The replicon plasmids constructed in our laboratory for WNV [9] and the DENV4 replicon described here are arranged in the order of 5′-untranslated region (UTR), the N-terminal coding sequence of capsid (C), Renilla luciferase (Rluc) reporter gene with a translation termination codon, and an internal ribosome entry site (IRES) element from encephalomyocarditis virus (EMCV) for cap-independent translation of the downstream ORF that codes for a polyprotein precursor, C_terE-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, followed by the 3′-UTR. In the second DENV4 replicon, the Rluc gene is fused sequentially downstream to the 20 amino acid (aa) FMDV 2A protease coding sequence, neomycin resistance gene (Neo^r), a termination codon, and the EMCV leader followed by the same polyprotein coding sequence and 3′-UTR as in the first replicon. The first replicon is useful to study by transient transfection experiments the cis-acting elements and trans-acting factors involved in viral RNA replication. The second DENV4 replicon is used to establish a stable monkey kidney (Vero) cell line by transfection of replicon RNA and selection in the presence of the G418, an analog of neomycin. This replicon is useful for screening and identifying antiviral compounds that are potential inhibitors of viral replication.