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      Zika Virus Replicons for Drug Discovery

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          Abstract

          The current epidemic of Zika virus (ZIKV) has underscored the urgency to establish experimental systems for studying viral replication and pathogenesis, and countermeasure development. Here we report two ZIKV replicon systems: a luciferase replicon that can differentiate between viral translation and RNA synthesis; and a stable luciferase replicon carrying cell line that can be used to screen and characterize inhibitors of viral replication. The transient replicon was used to evaluate the effect of an NS5 polymerase mutation on viral RNA synthesis and to analyze a known ZIKV inhibitor. The replicon cell line was developed into a 96-well format for antiviral testing. Compare with virus infection-based assay, the replicon cell line allows antiviral screening without using infectious virus. Collectively, the replicon systems have provided critical tools for both basic and translational research.

          Highlights

          • •

            A Zika virus replicon has been developed to study antivirals and to differentiate between viral translation and replication.

          • •

            A cell line carrying Zika virus replicon has been developed to screen and characterize antiviral inhibitors.

          Experimental systems are urgently needed to study Zika virus and to develop countermeasures. In this communication, we report the development of Zika virus replicon systems that can be used to study viral replication and pathogenesis as well as to screen and characterize antiviral inhibitors.

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          Most cited references22

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          Efficient initiation of HCV RNA replication in cell culture.

          K. J. Blight, A A Kolykhalov, C M Rice (2000)
          Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.
          Article 0 comments Cited 199 times – based on 0 reviews     Review now
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            Conserved elements in the 3' untranslated region of flavivirus RNAs and potential cyclization sequences.

            E. Strauss, Molly C. Pellitteri Hahn, Bonita Lee … (1987)
            We have isolated a cDNA clone after reverse transcription of the genomic RNA of Asibi yellow fever virus whose structure suggests it was formed by self-priming from a 3'-terminal hairpin of 87 nucleotides in the genomic RNA. We have also isolated a clone from cDNA made to Murray Valley encephalitis virus RNA that also appears to have arisen by self-priming from a 3'-terminal structure very similar or identical to that of yellow fever. In addition, 3'-terminal sequencing of the S1 strain of dengue 2 RNA shows that this RNA is also capable of forming a 3'-terminal hairpin of 79 nucleotides. Furthermore, we have identified two 20-nucleotide sequence elements which are present in the 3' untranslated region of all three viruses; one of these sequence elements is repeated in Murray Valley encephalitis and dengue 2 RNA but not in yellow fever RNA. In all three viruses, which represent the three major serological subgroups of the mosquito-borne flaviviruses, the 3'-proximal conserved sequence element, which is found immediately adjacent to the potential 3'-terminal hairpin, is complementary to another conserved domain near the 5' end of the viral RNAs, suggesting that flavivirus RNAs can cyclize (calculated delta G less than -11 kcal; 1 kcal = 4.184 kJ).
            Article 0 comments Cited 70 times – based on 0 reviews     Review now
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              Identification of cis-acting elements in the 3'-untranslated region of the dengue virus type 2 RNA that modulate translation and replication.

              Mariya Shapiro, Barry Falgout, M. Pilar Manzano … (2011)
              Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3'-untranslated region of the dengue virus (DENV) RNA can form two dumbbell structures (5'- and 3'-DBs) of unequal frequencies of occurrence. These structures have the propensity to form two potential pseudoknots between identical five-nucleotide terminal loops 1 and 2 (TL1 and TL2) and their complementary pseudoknot motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon RNA encoding the Renilla luciferase reporter indicated that all four motifs and the conserved sequence 2 (CS2) element within the 3'-DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect in translation, but translation is reduced by ∼60% in the TL1/TL2 double mutant, indicating that TL1 exhibits a cooperative synergy with TL2 in translation. Despite the variable contributions of individual TL and PK motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a noncanonical pathway. Taken together, our results indicate that the cis-acting RNA elements in the core region of DENV2 RNA that include two DB structures are required not only for RNA replication but also for optimal translation.
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                Author and article information

                Contributors
                Pei-Yong Shi
                Journal
                Journal ID (nlm-ta): EBioMedicine
                Journal ID (iso-abbrev): EBioMedicine
                Title: EBioMedicine
                Publisher: Elsevier
                ISSN (Electronic): 2352-3964
                Publication date PMC-release: 14 September 2016
                Publication date Collection: October 2016
                Publication date (Electronic): 14 September 2016
                Volume: 12
                Pages: 156-160
                Affiliations
                [a ]Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA
                [b ]Department of Pharmacology & Toxicology, Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA
                [c ]College of Animal Science and Technology, Southwest University, Chongqing, China
                [d ]Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, University of Leuven, Leuven, Belgium
                Author notes
                [* ]Corresponding author at: Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA.Department of Biochemistry & Molecular BiologyUniversity of Texas Medical BranchGalvestonTXUSA peshi@ 123456utmb.edu
                Article
                Publisher ID: S2352-3964(16)30420-0
                DOI: 10.1016/j.ebiom.2016.09.013
                PMC ID: 5078599
                PubMed ID: 27658737
                SO-VID: 4b954f55-5478-46ef-b16a-4ad97076bee3
                Copyright © © 2016 The Authors
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 31 August 2016
                Date revision received : 9 September 2016
                Date accepted : 13 September 2016
                Categories
                Subject: Research Paper

                Keywords: zika,drug discovery,replicon,flavivirus
                Data availability:
                Keywords: zika, drug discovery, replicon, flavivirus

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