Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing

Ng, Choon Ta; Gu, Feng; Phong, Wai Yee; Chen, Yen-Liang; Lim, Siew Pheng; Davidson, Andrew D.; Vasudevan, Subhash G.

doi:10.1016/j.antiviral.2007.06.007

Cited by 87 publications

(81 citation statements)

References 24 publications

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“…The culture supernatant obtained from BHK-21 cells at 72 h after infection with DENV-1 (multiplicity of infection [MOI] ＝ 0.1) was subjected to plaque assays. The result obtained was similar to the result of previous studies (15,25) as well as that obtained with the DNA-based replicon (Fig. 2B).…”

supporting

confidence: 82%

“…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)(9)(10)(11)(12)(13)(14)(15). The incorporation of firefly and Renilla luciferase reporter genes into DENV replicon constructs has facilitated the study of this virus.…”

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confidence: 99%

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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

et al. 2014

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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...

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confidence: 82%

mentioning

confidence: 99%

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

et al. 2014

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“…These designs ensured correct protein processing of all NS proteins that are largely involved in viral replication, as well as intact 5′ and 3′NCRs of the DENV-2 genome, thereby retaining essential structures/elements for initial viral protein translation and viral RNA replication. Successful establishment of subgenomic flavivirus replicons including WNV, YFV and DENV-2 have been demonstrated (Shi et al, 2002;Jones et al, 2005;Ng et al, 2007), but most of them are RNA-launched system. DNA-launched subgenomic DENV-2 replicons based on NGC strain has been achieved before (Pang et al, 2001), but it did not include any reporter gene.…”

Section: Discussionmentioning

confidence: 99%

“…For example, subgenomic WNV replicon expressing reporter gene was developed to promote antiviral drug screening (Lo et al, 2003a;Rossi et al, 2005;Shi et al, 2002). DENV-2 New Guinea C replicons containing GFP or luciferase reporter stabilized in BHK-21 cells were established for antiviral screening and siRNA testing (Ng et al, 2007). DENV-2 16681 replicon containing luciferase reporter was created and used to study 3′NCR structure and an anti-viral compound targeting the 3′NCR (Alvarez et al, 2005;Holden et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

et al. 2012

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Insertion of green fluorescent protein (GFP) encoding-gene into virus genes has provided a valuable tool for flavivirus research. This study aimed to develop dengue virus (DENV) replicons expressing GFP reporter that would provide a fast in vitro system to analyze functional roles of specific DENV sequences in viral replication. Two classes of recombinant replicon constructs were generated; one was a RNA-launched replicon with a GFP gene directly inserted into a fulllength DENV genome (FL-DENV/GFP), and the other consisted of 4 types of DNA-launched DENV subgenomic replicons with GFP replacement at various structural genes (Δ-DENV/GFP). The FL-DENV/GFP resulted in GFP expression in transfected cells with no viable DENV being recovered from the transfection. The Δ-DENV/GFP constructs with partial structural gene deletion (ΔC-, ΔCprM/M-, ΔprM/M-, or ΔE-) expressed bright and long lasting GFP. The GFP expression intensity in living cells correlated well with the level of RNA replication. Various mutations in the 5′noncoding region of DENV-2 previously shown to be important genetic determinants for virus replication and mouse virulence were incorporated into the 5 different replicon constructs. Characterizations of 29 mutants demonstrated that these replicons can provide a useful platform for a quick and powerful in vitro system to analyze genetic determinants of DENV replication. These constructs can also be useful for development of vectors expressing foreign genes for various researches.

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“…In addition, the expression of viral structural proteins in cells harbouring replicon RNA has been shown to produce single-round infectious particles (SRIPs), which are infectious, but progeny viruses cannot be spread from the infected cells, as the packaged genome lacks structural protein genes (Gehrke et al, 2003;Jones et al, 2005;Khromykh et al, 1998;Ng et al, 2007;Scholle et al, 2004;Yun et al, 2009). Furthermore, trans-packaging of replicons by the prM-E proteins from heterologous flaviviruses have been reported (Ansarah-Sobrinho et al, 2008;Yoshii et al, 2008).…”

mentioning

confidence: 99%

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon

Suzuki

Ishikawa

Konishi

et al. 2014

Journal of General Virology

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A method for rapid production of single-round infectious particles (SRIPs) of flavivirus would be useful for viral mutagenesis studies. Here, we established a DNA-based production system for SRIPs of flavivirus. We constructed a Japanese encephalitis virus (JEV) subgenomic replicon plasmid, which lacked the C-prM-E (capsid-pre-membrane-envelope) coding region, under the control of the cytomegalovirus promoter. When the JEV replicon plasmid was transiently cotransfected with a JEV C-prM-E expression plasmid into 293T cells, SRIPs were produced, indicating successful trans-complementation with JEV structural proteins. Equivalent production levels were observed when C and prM-E proteins were provided separately. Furthermore, dengue types 1-4, West Nile, yellow fever or tick-borne encephalitis virus prM-E proteins could be utilized for production of chimaeric flavivirus SRIPs, although the production was less efficient for dengue and yellow fever viruses. These results indicated that our plasmid-based system is suitable for investigating the life cycles of flaviviruses, diagnostic applications and development of safer vaccine candidates.

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Construction and characterization of a stable subgenomic dengue virus type 2 replicon system for antiviral compound and siRNA testing

Cited by 87 publications

References 24 publications

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

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

Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication

Production of single-round infectious chimeric flaviviruses with DNA-based Japanese encephalitis virus replicon

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