Development of a Novel Dengue-1 Virus Replicon System Expressing Secretory <i>Gaussia</i> luciferase for Analysis of Viral Replication and Discovery of Antiviral Drugs

Abstract

SUMMARY: Replicon systems have been used for high-throughput screening of anti-dengue virus (anti-DENV) inhibitors and for understanding mechanisms of viral replication. In the present study, we constructed novel DENV-1 replicons encoding Gaussia luciferase that was secreted into the culture medium. Two types of constructs were generated: RNA-based and DNA-based. Each type was translated in an internal ribosome entry site (IRES)-dependent or IRES-independent manner. Among these constructs, the DNA-based replicon employing IRES-dependent translation (DGL2) produced the highest titer. Luciferase levels in the culture medium revealed that the DGL2 replicon was inhibited by ribavirin (a well-known DENV inhibitor) at levels similar to those measured for drug inhibition of multi-round DENV-1 infection. These results indicate that the DNA-based IRES-driven DENV-1 replicon may facilitate studies on viral replication and antiviral compound discovery.Dengue virus (four serotypes [DENV-1-4]), which belongs to the genus Flavivirus of the family Flaviviridae, is transmitted to humans by Aedes mosquitoes and is the etiologic agent of dengue fever and dengue hemorrhagic fever (1). DENV annually infects 50-100 million humans in tropical and sub-tropical regions, posing a considerable public health threat in over 100 countries (2). However, at present, no specific antiviral drug or licensed vaccine is available for DENV infection (3). Therefore, the development of effective prophylactic and therapeutic measures is urgently required. The virus is an enveloped RNA virus with a single-stranded positive-sense genome of approximately 11 kb. A single long open reading frame (ORF) encodes a polyprotein that is processed by cellular and viral proteases into 3 structural (C, prM, and E) and 7 non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The structural proteins form the virus particle, whereas the NS proteins are involved in the replication of the viral genome.A subgenomic replicon is a self-replicating segment of the viral genome that lacks the genes encoding structural proteins. In general, a subgenomic replicon is established by transfection of in vitro transcribed RNA. Recently, DNA-based replicon systems, in which transcription is controlled by a cytomegalovirus (CMV) promoter, have been developed from alphavirus, arterivirus, and flavivirus (4-7). The DNA-based replicons are more stable than RNA-based constructs and the replicons can be directly transfected (thus without in vitro transcription) (5,6). Reporter replicon systems have been used for the analysis of flavivirus replication mechanisms and for conducting high-throughput screening for antiviral compounds (8-15). The incorporation of firefly and Renilla luciferase reporter genes into DENV replicon constructs has facilitated the study of this virus. Recently, genes encoding secretory luciferases have been used in studies on other viruses such as retrovirus, togavirus, herpesvirus, and flavivirus (16)(17)(18)(19).To generate a more useful reporter replic...

Interferon-stimulated TRIM69 interrupts dengue virus replication by ubiquitinating viral nonstructural protein 3

“…Consistently, the released viruses in cell supernatants were also decreased in TRIM69 overexpressed cells ( Fig 2E ). In addition, we also used a luciferase-based DENV replicon (DGL2), derived from DENV-1 [ 51 , 52 ], to analyze the function of TRIM69 on virus replication. The replicon replication was also impaired by TRIM69 ectopic expression ( Fig 2F ).…”

“…All NS genes were amplified from cDNA of DENV-2 NGC strain. A DNA-based DENV replicon DGL2 (for DENV type 1) was generously provided by Dr. Takayuki Hishiki (Kyoto University, Kyoto, Japan) [ 51 ].…”

Hirsutine, an Indole Alkaloid of Uncaria rhynchophylla, Inhibits Late Step in Dengue Virus Lifecycle

“…To further clarify the mechanism of hirsutine-mediated suppression of DENV replication, we performed a reporter subgenomic replicon assay. The transient replicon system is a useful tool for estimating the stage of action ( Figure 3A ; Kato et al, 2014 ). After 24 h and 72 h of replicon plasmid transfection, the Gaussia luciferase activity in the culture supernatant was analyzed.…”

“…Furthermore, the replicon assay showed that hirsutine did not restrict the viral genome translation and replication steps. Because our subgenome replicon, DGL2, deleted almost the entire structural region of DENV-1, the assay could specifically analyze the viral genome translation and replication steps ( Kato et al, 2014 ). Hence, the replicon system is a useful tool for screening antiviral reagents and for elucidating the mechanism of action in the virus lifecycle ( Kato and Hishiki, 2016 ).…”

“…A transient replicon assay was conducted using DGL2 and DGL2-mut as described previously ( Kato et al, 2014 ). A549 cells were cultured in a 24-well plate and transfected with DGL2 and DGL2-mut using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland); 10 μM of hirsutine was added at baseline.…”