Fig 1.
Luciferase assay for miRNAs to CHIKV 3′ UTR binding.
(A) Sequence of CHIKV 3' UTR cloned into the pmirGLO vector, and miRNA-3'UTR binding with mutations on boxed sequence of 3'UTR, where sequence was replaced with nucleotide Adenine. (B) Relative expression of miRNAs miR-2944b-5p and miR-2b cloned into pmR-mCherry, at 12hrs and 24hrs post transfection into HEK-293T. (C) Luciferase assay for miR-2b and miR-2944b-5p binding to 3' UTR. (D) Luciferase assay showing binding of miR-2b and miR-2944b-5p to 3' UTR with their mutated (boxed sequence of seed region) binding site. Empty pmirGLO vector along with miR-2944b-5p cloned in pmR-mCherry vector was taken as control. The experiments were repeated at least thrice and each experiment included at least three treatments Data are expressed as mean ± SEM, *P < 0.05, ****P < 0.0001.
Fig 2.
Effect of miRNA on viral replication using qRT-PCR.
(A) Expression level of CHIKV genomic RNA after 24 hrs post infection (MOI 1) and 48 hrs post antagomir transfection for miR-2944b-5p and miR-2b, relative to uninfected cells. The mock transfected contained scrambled miRNA. (B) QRT-PCR analysis of CHIKV genomic RNA in Ae. aegypti mosquitoes under different conditions (CHIKV infection, CHIKV infection with scrambled miRNA (mock), and CHIKV infection with Antimir-2944b-5P) and at different time points, 24hrs, 48hrs, and 72hrs post infection. The experiments were repeated at least thrice and each experiment included at least three treatments Data are expressed as mean ± SEM,**P < 0.01 ***P < 0.001.
Fig 3.
Ae. aegypti cellular target prediction and validation.
(A) List of top 28 predicted cellular targets for miR-2944b-5p using RNAhybrid tool. (B) Luciferase assay shows relative percentage of luciferase/renilla luminescence for miR-2944b-5p binding to its three targets (AAEL011195, AAEL008432, and AAEL010484), Empty pmirGLO vector along with miR-2944b-5p cloned in pmR-mCherry vector was taken as control (C) Binding region between vps-13 and miR-2944b-5p. (D) Relative expression of vps-13 at different time points 24hrs, 48hrs, 72hrs during miR-2944b-5p inhibition in Aag2 cell line using Antimir (E) Relative expression of vps-13 at different time points 24hrs, 48hrs, 72hrs during miR-2944b-5p inhibition in Ae. aegypti using Antimir. The experiments were repeated at least thrice and each experiment included at least three treatments. Data are expressed as mean ± SEM, *P < 0.05.
Fig 4.
vps-13 and CHIKV infection in Aag2 cells.
(A) Relative expression vps-13 during CHIKV infection at different time points, 24hrs, 48hrs and 72hrs in Aag2 cell line and Ae. aegypti mosquito. (B) Relative expression of vps-13 upon dsRNA transfection at different time points 24hrs, 48hrs and 72hrs in Aag2 cell line and (C) Ae. aegypti mosquito, mock contained the gfp dsRNA as negative control. (D) Relative expression of viral genomic RNA in Aag2 cell line and (E) Ae. aegypti mosquito upon vps-13 inhibition through dsRNA at 24hrs post infection (cells were infected after 24hrs of dsRNA transfection and mock dsRNA contained gfp dsRNA as a negative control). The experiments were repeated at least thrice and each experiment included at least three treatments. Data are expressed as mean ± SEM, ****P < 0.0001, **P < 0.01, *P < 0.05.
Fig 5.
Effect of simultaneous inhibition of miR-2944b-5p and vps-13 during CHIKV infection in Aag2 cells.
The experiments were repeated at least thrice and each experiment included at least three treatments. Data are expressed as mean ± SEM, **P < 0.01, *P < 0.05.
Fig 6.
Effect of CHIKV, Antimir-2944b-5p and vps-13 on MMP in the Aag2.
(A) The MMP in the Aag2 cell line. After 24 hrs post transfections (with Antimir-2944b-5p and vps-13 dsRNA) cells were infected with CHIKV (MOI 1). After 24 hrs post infection, the cells were stained with the mitochondrial-selective JC-1 dye for mitochondrial potential and DAPI to stain nuclei for confocal imaging analysis. (B) The ratio of JC-1 red and JC-1 green fluorescence was measured and represented as histogram. The experiments were repeated at least twice and each experiment included at least three treatments. ***P < 0.001, *P < 0.05.
Fig 7.
Proposed model for the miR-2944b-5p interplay between CHIKV genome and mitochondrial integrity.