title: Suppression of the Arboviruses Dengue and Chikungunya Using a Dual-Acting Group-I Intron Coupled with Conditional Expression of the Bax C-Terminal Domain.
abstract: In portions of South Asia, vectors and patients co-infected with dengue (DENV) and chikungunya (CHIKV) are on the rise, with the potential for this occurrence in other regions of the world, for example the United States. Therefore, we engineered an antiviral approach that suppresses the replication of both arboviruses in mosquito cells using a single antiviral group I intron. We devised unique configurations of internal, external, and guide sequences that permit homologous recognition and splicing with conserved target sequences in the genomes of both viruses using a single trans-splicing Group I intron, and examined their effectiveness to suppress infections of DENV and CHIKV in mosquito cells when coupled with a proapoptotic 3' exon, ΔN Bax. RT-PCR demonstrated the utility of these introns in trans-splicing the ΔN Bax sequence downstream of either the DENV or CHIKV target site in transformed Aedes albopictus C6/36 cells, independent of the order in which the virus specific targeting sequences were inserted into the construct. This trans-splicing reaction forms DENV or CHIKV ΔN Bax RNA fusions that led to apoptotic cell death as evidenced by annexin V staining, caspase, and DNA fragmentation assays. TCID50-IFA analyses demonstrate effective suppression of DENV and CHIKV infections by our anti-arbovirus group I intron approach. This represents the first report of a dual-acting Group I intron, and demonstrates that we can target DENV and CHIKV RNAs in a sequence specific manner with a single, uniquely configured CHIKV/DENV dual targeting group I intron, leading to replication suppression of both arboviruses, and thus providing a promising single antiviral for the transgenic suppression of multiple arboviruses.
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: James R
authors.initials: JR
authors.lastname: Carter
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Samantha
authors.initials: S
authors.lastname: Taylor
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Tresa S
authors.initials: TS
authors.lastname: Fraser
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Kucharski
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: James L
authors.initials: JL
authors.lastname: Dawson
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana, United States of America.
authors.forename: Malcolm J
authors.initials: MJ
authors.lastname: Fraser
mesh_terms: D000330:Aedes / Q000166:cytology / Q000821:virology*; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002646:Chikungunya virus / Q000235:genetics* / Q000378:metabolism; D053938:DNA Fragmentation; D003716:Dengue Virus / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology / Q000821:virology; D005091:Exons; D005260:Female; D006801:Humans; D007438:Introns; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000737:chemistry / Q000235:genetics; D020040:Trans-Splicing; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D017304:Annexin A5; C494826:BAX protein, human; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D051028:bcl-2-Associated X Protein; D020169:Caspases