Targeting of highly conserved Dengue virus sequences with anti-Dengue virus trans-splicing group I introns

Abstract

BackgroundDengue viruses (DENV) are one of the most important viral diseases in the world with approximately 100 million infections and 200,000 deaths each year. The current lack of an approved tetravalent vaccine and ineffective insecticide control measures warrant a search for alternatives to effectively combat DENV. The trans-splicing variant of the Tetrahymena thermophila group I intron catalytic RNA, or ribozyme, is a powerful tool for post-transcriptional RNA modification. The nature of the ribozyme and the predictability with which it can be directed makes it a powerful tool for modifying RNA in nearly any cell type without the need for genome-altering gene therapy techniques or dependence on native cofactors.ResultsSeveral anti-DENV Group I trans-splicing introns (αDENV-GrpIs) were designed and tested for their ability to target DENV-2 NGC genomes in situ. We have successfully targeted two different uracil bases on the positive sense genomic strand within the highly conserved 5'-3' cyclization sequence (CS) region common to all serotypes of DENV with our αDENV-GrpIs. Our ribozymes have demonstrated ability to specifically trans-splice a new RNA sequence downstream of the targeted site in vitro and in transfected insect cells as analyzed by firefly luciferase and RT-PCR assays. The effectiveness of these αDENV-GrpIs to target infecting DENV genomes is also validated in transfected or transformed Aedes mosquito cell lines upon infection with unattenuated DENV-2 NGC.ConclusionsAnalysis shows that our αDENV-GrpIs have the ability to effectively trans-splice the DENV genome in situ. Notably, these results show that the αDENV-GrpI 9v1, designed to be active against all forms of Dengue virus, effectively targeted the DENV-2 NGC genome in a sequence specific manner. These novel αDENV-GrpI introns provide a striking alternative to other RNA based approaches for the transgenic suppression of DENV in transformed mosquito cells and tissues.

Computational prediction of efficient splice sites for trans-splicing ribozymes

“…These ribozymes could be used to treat genetic disorders by repairing the sequence of a mutated mRNA (Sullenger and Cech 1994;Lan et al 1998;Phylactou et al 1998), to selectively kill cancer cells by splicing a sequence that encodes a toxic peptide into cancer-specific mRNAs Kwon et al 2005;Lee et al 2010), and to kill virally infected cells with the same strategy Carter et al 2010).…”

Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence

“…DENV-2 replication in mosquito cell cultures was suppressed by group 1 splicing introns targeted to the 5ЈCS region (8), and WNV and St. Louis encephalitis virus infections were inhibited in vitro and WNV infection was inhibited in a mouse model by a 5ЈCS-targeting siRNA conjugated to a 41-mer peptide (73). We observed a potent inhibition of YFV and a modest inhibition of WNV by the targeting of their respective 5ЈCS regions.…”

Disruption of dengue virus transmission by mosquitoes

“…Initially, a GrpI was identified in the ciliate protozoa Tetrahymena thermophila as a self-splicing intron [76]. Since then GrpI have been used for various applications such as repair of defective mRNA transcripts or as antiviral effectors targeting HIV-1, cucumber mosaic virus, HCV, or DENV1–4 [77–79,80**,81**]. A GrpI consists of an external (EGS) and an internal guide sequence (IGS), a trans-splicing domain, and a 3′ exon-of-choice (which can be derived from a pro-apoptotic gene) (Figure 2).…”

“…Carter et al [80**] designed six different GrpI targeting the highly conserved cyclization (CS) sequences (5′CS, CS1, and CS2) in the genomes of DENV1–4. In vitro assays revealed that two of the six GrpI, αDENV-U143-FL and αDENV-U134-FL, targeting U143 and U134 respectively, of the DENV2 5′CS were highly effective, with αDENV-U143-FL being able to cleave the viral RNA of all four DENV serotypes, whereas αDENV-U134-FL was specific to DENV2.…”

“…DENV2 infection of GrpI transformed C6/36 cells resulted in significantly increased luciferase activity in comparison to the control. Importantly, DENV2 titers in cells expressing αDENV-U143-FL and αDENV-U143-FL were significantly reduced [80**]. Eventually, the luciferase encoding 3′ exon was replaced with an exon encoding ∆N bax, a truncated version of the pro-apoptotic gene tbax showing increased pro-apoptotic potency [82].…”