Hammerhead Ribozyme Mediated Suppression of Chikungunya Virus Replication in Vero Cells

Chikungunya (CHIKV) virus is an alphavirus, transmitted to humans by the bite of the infected Aedes mosquitoes. Transgenic mosquitoes with resistance to CHIKV replication could control the spread of the virus among humans. We designed 8 hammerhead ribozymes (hRz#1, 2, 3, 4, 5, 6, 7, 8) against conserved regions within the nonstructural genes (nsp1, nsp2, nsp3 and nsp4) of the CHIKV viral genome. In vitro cleavage assays demonstrated the catalytic activity of these hRzs against artificial CHIKV target RNAs. These hRzs were cloned into a pantrophic retrovirus vector with Aedes tRNAval pol III driving expression of the hRzs, and a poly A (60) tail sequence to recruit RNA helicase for disruption of secondary structure in the target viral RNA. The cloned plasmids were transfected into Vero cells and selected for hygromycin resistance for 6-8 weeks. Reverse transcriptase PCR confirmed expression of all hRzs in the transformed cells. The transformed cell lines were challenged with the CHIKV 181/25 strain at a MOI of 0.0001 and the viral titer of the supernatants was determined at 2 days post infection by using a TCID50-CPE assay. hRz#1 and hRz#8 targeting the nsp1 and nsp4 genes, respectively, yielded 2 and 5 logs suppression of CHIKV replication as compared to wild type infected Vero cells. Homogenous cell populations were obtained by single cell sorting of hRz transformed cells using FACS, and the clones were challenged with CHIKV at a MOI of 0.0001. The viral titers of supernatants determined at 3 dpi using TCID50-IFA demonstrated complete of suppression of CHIKV replication with some of the hRz-transformed Vero cell clones expressing hRz# 2, 4, 5, 6 and 8. QRT-PCR also showed significant reduction of CHIKV RNA copies with these hRz-transformed Vero cell clones as compared to the control negatives. Indirect assessment of caspase-3 activity from the infected supernatants collected from those clones revealed very low levels of caspase 3 activity as compared to negative control, CHIKV infected Vero cells. HRz# 2, 4, 5, 6 and 8 are ideal candidates for generating transgenic refractoriness in mosquitoes, and will be cloned into the pXLbacII 3xp3 ECFP piggyBac transposon vector for injection of mosquito embryos. Based on these in vitro studies, we anticipate that transgenic mosquitoes expressing these hammerhead ribozymes will be refractory to CHIKV infection and transmission, and may be useful in effective suppression of CHIKV in natural populations.