Characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus

Yang, Chi; Tsai, Ming Han; Hu, Han Shu; Pu, Szu-Yuan; Wu, Ren Huang; Wu, Szu Huei; Lin, Hui Mei; Song, Jen Shin; Chao, Yu Sheng; Yueh, Andrew

doi:10.1016/j.antiviral.2013.03.001

Cited by 30 publications

(44 citation statements)

References 61 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To determine whether the decreased infectivity of the various NS2A mutants resulted from defects in viral RNA replication, alanine substitu-tion mutations were introduced into either a DNA-launched DENV2 replicon plasmid (pCMV-DV2Rep) (Fig. 4A) or an RNA-launched DENV2 replicon plasmid (DV2Rep) (30). Transcription of the replicon RNA derived from the DNA-launched replicon was driven by the CMVmin promoter, and the integrity of the 3= end was preserved by using an inverted HDV ribozyme sequence.…”

Section: Resultsmentioning

confidence: 99%

Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

Tsai

et al. 2017

J Virol

Self Cite

View full text Add to dashboard Cite

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMS1 to -8) and participates in RNA replication, virion assembly, and host antiviral response. However, the roles of specific amino acid residues within the pTMS regions of NS2A during the viral life cycle are not clear. Here, we explore the function of DENV NS2A by introducing a series of alanine substitutions into the N-terminal half (pTMS1 to -4) of the protein in the context of a DENV infectious clone or subgenomic replicon. Six NS2A mutants (NM5, -7, -9, and -17 to -19) around pTMS1 and -2 displayed a novel phenotype showing a Ͼ1,000-fold reduction in virus yield, an absence of plaque formation despite wild-type-like replicon activity, and infectious-virus-like particle yields. HEK-293 cells infected with the six NS2A mutant viruses failed to cause a virus-induced cytopathic effect (CPE) by MitoCapture staining, cell proliferation, and lactate dehydrogenase release assays. Sequencing analyses of pseudorevertant viruses derived from lethal-mutant viruses revealed two consensus reversion mutations, leucine to phenylalanine at codon 181 (L181F) within pTMS7 of NS2A and isoleucine to threonine at codon 114 (I114T) within NS2B. The introduction of an NS2A-L181F mutation into the lethal (NM15, -16, -25, and -33) and CPE-defective (NM7, -9, and -19) mutants substantially rescued virus infectivity and virus-induced CPE, respectively, whereas the NS2B-L114T mutation rescued the NM16, -25, and -33 mutants. In conclusion, the results revealed the essential roles of the N-terminal half of NS2A in RNA replication and virus-induced CPE. Intramolecular interactions between pTMSs of NS2A and intermolecular interactions between the NS2A and NS2B proteins were also implicated. IMPORTANCEThe characterization of the N-terminal (current study) and C-terminal halves of DENV NS2A is the most comprehensive mutagenesis study to date to investigate the function of NS2A during the flaviviral life cycle. A novel region responsible for virus-induced cytopathic effect (CPE) within pTMS1 and -2 of DENV NS2A was identified. Revertant genetics studies implied unexpected relationships between various pTMSs of DENV NS2A and NS2B. These results provide comprehensive information regarding the functions of DENV NS2A and the specific amino acids and transmembrane segments responsible for these functions. The positions and properties of the rescuing mutations were also revealed, providing important clues regarding the manner in which intramolecular or intermolecular interactions between the pTMSs of NS2A and NS2B regulate virus replication, assembly/secretion, and virusinduced CPE. These results expand the understanding of flavivirus replication. The

show abstract

Section: Resultsmentioning

confidence: 99%

Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

Tsai

et al. 2017

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…in Dengue virus [17]. Our technique can complement current approaches using ordinary differential equations and can help overcome problems of parameter identifiability and correlation of parameters (like the lifetime of infected cells) [5].…”

Section: Discussionmentioning

confidence: 92%

“…This suggests a novel way to combine traditional differential equation models with models fit to replicon data and could be applied to other emerging diseases, e.g. Dengue virus for which replicons have also been constructed [17].…”

Section: Discussionmentioning

confidence: 99%

A Computational Technique to Estimate Within-Host Productively Infected Cell Lifetimes in Emerging Viral Infections

Banerjee¹

2017

Interdiscip. Descr. Complex Syst.

View full text Add to dashboard Cite

Emerging viruses cause a lot of fatalities as they jump to humans from other species. Here we develop a novel technique to computationally estimate an important parameter of within-host viral infection: the lifespan of infected cells. Our approach is general and can be applied to a large class of viruses and leverages experimental data from replicon studies. Current techniques have difficulties reliably estimating infected cell lifetimes due to parameter identifiability and correlation of parameters. The infected cell lifetime is an important parameter that gives an estimate of how fast virus levels will decline. Our method would also help determine if some infected cells are short-lived or have longer lifespans with the consequence that longer lived cells could be reservoirs of infection. This would give a mechanistic understanding of why particular cell types are reservoirs of infection and may motivate therapy targeted towards these cell types. We apply our technique to West Nile virus (WNV), an emerging disease of public health relevance related to Zika virus. Our analysis suggests that the most abundant infectible cells are short-lived and could motivate therapy that targets these particular cells. Our approach is very general and can be combined with more traditional methods of using differential equation models to simulate viremia in hosts: the combination of these two techniques will likely yield results that may not be achievable using the models in isolation. This will be of great interest especially in modelling emerging diseases.

show abstract

“…Similarly, Qing Min et al (16) also performed antiviral compound analysis through the infection of Vero and Huh-7 cell lines with dengue. DENV replicon constructs showed higher rate of replication in human Huh7.5 cells than in Chinese hamster BHK21 cells (1). A study was carried out to explore anti dengue effects in plants (26).…”

Section: Discussionmentioning

confidence: 99%

“…Dengue virus (DENV) is transmitted to human by Aedes mosquitoes; it causes dengue fever (DF) and dengue hemorrhagic fever (DHF), which is a self-limiting febrile illness (1,2). Dengue fever is relatively mild, but DHF leads to the life-threatening dengue shock syndrome (DSS).…”

Section: Introductionmentioning

confidence: 99%

Antiviral Activity of Thiazolide Derivatives Against Dengue Virus in Huh-7 Cell Line

Yasmin

Yaqub

Khan

et al. 2017

Jundishapur J Microbiol

View full text Add to dashboard Cite

Background: Dengue is one of the main health problems worldwide with dramatic increases in reported cases in the last few decades. Now, dengue is a major threat to half of the world's population and approximately 50 million infections occur every year. Objectives: The major aim of this study was to screen out different synthetic antiviral compounds against dengue virus. As treatments used for dengue virus infections are nonspecific and effectless, it is necessary to find specific antiviral compounds that may be helpful in the treatment of dengue fever. In cell culture models, thiazolides are active against anaerobic bacteria, protozoa, and a range of viruses. Methods: Samples from positive dengue fever patients were included in the study. For dengue serotype-2, serum samples were further confirmed by serotyping protocol. Results: After toxicological analysis, a sharp significant antiviral response was shown by compounds A and B decreasing the viral titer of serotype 2 as 46% and 53%, respectively. A greater antiviral activity was indicated by compound "B" against NS3 gene as compared to compound "A". Against NS3 gene, a comparable antiviral activity was observed for compounds "A" and "B". Conclusions: From our results, it can be concluded that compounds A and B (Thiazolides) as dengue antiviral drugs can reduce viral load in patients if their inhibitory effects are reproduced in vivo. To identify the specific active ingredients, further studies are required by using various techniques to determine the efficacy of these compounds in animal model.

show abstract

Characterization of an efficient dengue virus replicon for development of assays of discovery of small molecules against dengue virus

Cited by 30 publications

References 61 publications

Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

Mutagenesis of Dengue Virus Protein NS2A Revealed a Novel Domain Responsible for Virus-Induced Cytopathic Effect and Interactions between NS2A and NS2B Transmembrane Segments

A Computational Technique to Estimate Within-Host Productively Infected Cell Lifetimes in Emerging Viral Infections

Antiviral Activity of Thiazolide Derivatives Against Dengue Virus in Huh-7 Cell Line

Contact Info

Product

Resources

About