title: Effective suppression of dengue virus using a novel group-I intron that induces apoptotic cell death upon infection through conditional expression of the Bax C-terminal domain.
abstract: BACKGROUND
  Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ΔN Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells.
  
  
  RESULTS
  Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ΔN Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-ΔN Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-ΔN Bax fusion protein expression induces apoptotic cell death.
  
  
  CONCLUSIONS
  This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ΔN Bax 3' exon that trans-splices conserved sequences of the 5' CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease.
authors.forename: James R
authors.initials: JR
authors.lastname: Carter
authors.forename: James H
authors.initials: JH
authors.lastname: Keith
authors.forename: Tresa S
authors.initials: TS
authors.lastname: Fraser
authors.forename: James L
authors.initials: JL
authors.lastname: Dawson
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Kucharski
authors.forename: Kate M
authors.initials: KM
authors.lastname: Horne
authors.forename: Stephen
authors.initials: S
authors.lastname: Higgs
authors.affiliation: Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, Indiana 46556, USA. fraser.1@nd.edu.
authors.forename: Malcolm J
authors.initials: MJ
authors.lastname: Fraser
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002460:Cell Line; D009033:Culicidae; D003715:Dengue / Q000821:virology; D003716:Dengue Virus / Q000145:classification / Q000235:genetics*; D005091:Exons; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors; D007438:Introns; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D065288:Serogroup; D020040:Trans-Splicing; D014779:Virus Replication / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics*
chemical_list: D051028:bcl-2-Associated X Protein