Figure 1.
Inhibition of luciferase activity by NP miRNA expressed from a mouse miR30-based lentiviral vector.
(a) Schematic diagram of the miR30-NP lentiviral vector. The backbone of the vector is pBL2. (b) Sequence of miR30-NP hairpin. Blue color letters represent the antisense sequences targeting influenza NP transcript. Flanking and hairpin sequences are miR30. (c) Inhibition of luciferase activity by transient transfection of miR30-NP lentiviral vector in DF-1 cells. Psicheck-2 dual luciferase reporter plasmid (50 ng) and miR30-NP lentiviral vector (450 ng) were co-transfected in DF-1 cells. Firefly and Renilla luciferase activities were measured 48 hrs later. Shown are relative Renilla to firefly luciferase activities after normalization to the ratio of control reporter plasmid without target sequence (set as 100%). (d) Inhibition of luciferase activity by stably integrated miR30-NP lentiviral vector in DF-1 cells. Cells were infected with lentivirus at an MOI of 0.1 then selected with puromycin and further sorted for GFP-positive cells (>95%). The transduced cells were then transfected with psicheck-2 dual luciferase reporter plasmid (50 ng) and a control plasmid pUC18 (450 ng). Luciferase activity was measured and normalized as in (c). All bar graphs represent means ± standard deviations (SD) of three independent experiments.
Figure 2.
Inhibition of luciferase activity by endogenous and lentiviral expressed chicken miRNAs.
(a) Inhibition of luciferase activity by lentiviral expressed chicken miRNAs. Endogenous chicken miRNAs plus ∼200 bp flanking sequences on both sides were cloned into pLB2 vector. The corresponding sense and antisense target sequences were cloned into psicheck-2 dual luciferase reporter plasmid. DF-1 cells were transfected with lentiviral vector (450 ng) and the corresponding reporter plasmid (50 ng) and luciferase activities were measured 48 hrs later. Shown are relative Renilla luciferase activities (means ± SD, n = 3) of chicken miRNA expressed from the sense (S) and antisense (AS) strands. (b) Inhibition of luciferase activity by endogenously expressed miRNAs. DF-1 cells were co-transfected with 450 ng pUC-18 (as transfection control) and 50 ng psicheck-2 reporter plasmids with endogenous miRNA target sequences and luciferase activities were measured as above. Shown are relative Renilla luciferase activities (means ± SD, n = 3) of sense (S) or antisense (AS) strand of miRNAs.
Figure 3.
Inhibition of luciferase activity by NP miRNA expressed from chicken miRNA-based lentiviral vectors.
(a) Structures and sequences of the miR21-NP and miR126-NP. Mature miR21 or miR126 sequences were replaced with anti-influenza NP sequences (blue). (b) Structures and sequences of miR21-NP-shRNA and miR126-NP-shRNA. Anti-influenza NP sequences were in blue. (c) Inhibition of luciferase activity by NP miRNA expressed from lentiviral vectors from a and b. DF-1 cells were co-transfected with lentiviral vector and the corresponding reporter plasmid, and luciferase activity was measured 48 hrs after transfection. Shown are average relative Renilla luciferase activities (n = 3). (d) Inhibition of luciferase activity by stably expressed NP miRNA. DF-1 cells were infected with miR126-NP lentivirus (MOI = 0.1) and were selected with puromycin until GFP-positive cells reached >95%. Cells were then transfected with reporter plasmid and luciferase activity was assayed 48 hrs later. Average Renilla luciferase activity is shown (n = 3).
Figure 4.
Inhibition of luciferase activity by multiple anti-influenza miRNAs expressed from chicken miR126-based lentiviral vectors.
(a) Schematic diagram of lentiviral vectors expressing one, two or three anti-influenza miRNAs. pLB2, lentiviral vector backbone; pLB2-NP, pLB2-PB1-NP, and pLB2-PB1-NP-PA, lentiviral vectors expressing NP miRNA, NP and PB1 miRNAs, and NP, PB1 and PA miRNAs, respectively. (b) Inhibition of luciferase activity by NP, PB1 or PA miRNAs in transient transfection assays. DF-1 cells were co-transfected with lentiviral vectors (450 ng) and corresponding psicheck-2 reporter plasmids (50 ng). Luciferase activity was measured 48 hrs later. Shown are relative Renilla luciferase activities (means ± SD, n = 3). (c) Inhibition of luciferase activity by NP, PB1 or PA miRNAs in stably transduced DF-1. DF-1 cells were transduced with pLB2-PB1-NP or pLB2-PB1-NP-PA lentivirus at an MOI of 0.1. Transduced cells (>95% GFP-positive) were transfected with reporter plasmid with NP, PB1 or PA target sequences. Luciferase activity was measured 48 hrs after transfection. Shown are relative Renilla luciferase activities (means ± SD, n = 3). (d–f) Detection of NP, PB1 and PA siRNAs by small RNA Northern blotting. Total RNA was isolated from DF-1 cells that were either transiently transfected with pLB2-PB1-NP-PA lentiviral vector or stably transduced with pLB2-PB1-NP-PA lentivirus. 6 µg RNA from transfected cells (lane 1) or 15 µg of RNA from stably transduced cells (lane 2) were fractionated by electrophoresis on a 15% poly-acrylamide gel. Following transfer, the blots were hybridized with NP, PB1 or PA specific probes and imaged. U6 RNAs were also probed as a loading standard. Molecular weight markers, U6 RNA, and NP, PB1 or PA siRNAs are indicated. Note, the processed PB1 miRNA ran slightly slower than NP and PA miRNAs.
Figure 5.
Inhibition of influenza virus production in transduced Vero cells expressing multiple artificial anti-influenza miRNAs.
Vero cells were transduced with pLB2-NP, pLB2-PB1-NP, pLB2-PB1-NP-PA, and CPGM (Control) lentiviruses and selected with puromycin to establish transduced cells (>95% GFP-positive). (a) The transduced cells were transfected with the indicated reporter plasmids to determine inhibition of luciferase activity. Shown are relative Renilla luciferase activities (means ± SD, n = 3). (b) Stably transduced cells were infected with PR8 virus at an MOI of 0.01. 48 hour after infection, the supernatants were collected and assayed for virus titer (PFU) by plaque assay on MDCK cells. Shown are average (means ± SD, n = 3) virus titers per milliliter supernatants. * P<0.001 by two way t-test.
Figure 6.
Inhibition of luciferase activity in non-chicken cells by miRNAs expressed from the miR126-based lentiviral vector.
(a) Inhibition of luciferase activity in 293T, MDCK, MEF and Vero cells. Cells were co-transfected with pLB2-PB1-NP-PA lentiviral vector (450 ng) and the appropriate reporter plasmid (50 ng). Luciferase activity was measured 48 hrs later. Shown are relative Renilla luciferase activities (means ± SD, n = 3). (b) Inhibition of luciferase activity by miR126-NP transcribed from the U6 promoter. DF-1 cells were co-transfected with either pLL3.7-NP or pLB2-NP lentiviral vector plus the reporter plasmid. Luciferase activity was assayed 48 hrs later. Shown are relative Renilla luciferase activities (means ± SD, n = 3).
Table 1.
Sequences of PCR primers for endogenous chicken miRNAs cloning.
Table 2.
Sequences of anti-influenza artificial miRNA cassettes.
Table 3.
Endogenous influenza sequences cloned into the psicheck-2 dual luciferase reporter as the targets for anti-influenza shRNAs and artificial miRNAs.