Figure 1.
In vitro transcription of inosine-containing RNA.
RNA was synthesized using Megascript in vitro transcription assay (Ambion, Austin, TX, USA) and pGEM-Luc vector (panel A) as template (Promega, Madison, WI). For RNA with inosine content, inosine tri-phosphate (Sigma, St. Louis, MO, USA) was added to the reaction mixture. Panel B, to quantify incorporation of inosines, RNA was digested by phosphodiesterase and was then separated by HPLC. Relative nucleoside incorporation was determined by calculating the area under each peak. The synthesized RNA is 0% (a), 6% (b), 10% (c) or 16% (d) inosines incorporations.
Figure 2.
Ino-RNA activates primary human cells.
Panel A, In vitro transcribed N-RNA and Ino-RNA (6%, 10% and 16% inosine content) were added directly to PHBE cells at 10 µg/ml final concentration. After 2 hr, total RNA was extracted and used in real-time RT-PCR (n≥4). We next performed a concentration curve using Ino-RNA with 10% inosine content (2 hr, panel B, n = 4) and kinetic studies (panel C) analysis of Ino-RNA induction of inflammatory cytokines (n = 4). Panel D, treatment of primary human alveolar macrophages with N-RNA and 10% Ino-RNA (n≥4). Error bars indicate standard error of the mean (± SEM, *p<0.05, **p<0.01, ***p<0.001).
Figure 3.
RSV replication is reduced by Ino-RNA treatment.
Panels A, PHBE cells, were treated with 10% Ino-RNA (20 µg/ml for), N-RNA (20 µg/ml), or IFN-α (1000 Units/ml) for 24 hr prior to RSV infection at 0.1 PFU/cell. Cells were collected after an additional 24 hr, for RNA extraction. Total RNA from infected cells were extracted for RT-PCR quantification of RSV-NS1 transcript (n≥3). Panel B, BEAS-2B cells were treated with 10% Ino-RNA (5, 10, 20 µg/ml), N-RNA (5,10, 20 µg/ml) (n≥3). Panel C, BEAS-2B cells were treated as above and after 48 hr infection with RSV at MOI of 0.1 PFU/cell, plaque assays were performed (n = 3). Error bars indicate standard error of the mean (± SEM, *p<0.05).
Figure 4.
Ino-RNA induces inflammatory responses in C57BL/6 mice.
Mice were intratracheally instilled with PBS, N-RNA or 10% Ino-RNA, and BAL fluid was harvested after 24 hr. Panel A, cytokine protein levels were determined in BAL fluid by ELISA (n = 3, ± SEM, *p<0.05, *** p<0.001). X-axis presents different RNA concentration. Panel B, flow cytometry analysis of neutrophil recruitment in BAL.
Figure 5.
Inosines incorporation increases secondary structures of RNA.
Panel A, N-RNA, 6%, 10% and 16% Ino-RNA were electrophoresed on non-denaturing or denaturing agarose gels in the absence or presence of MgCl2 and KCl. Panel B, RNA secondary structures were determined by acridine orange staining in the presence of MgCl2 and KCl followed by fluorescence spectral analysis. Panel C, Direct interaction of purified recombinant PKR with Ino-RNA was examined by in vitro kinase reaction followed by western blot analysis. Panel D, BEAS-2B cell extracts were used in in vitro kinase reaction in the presence of N-RNA (3 µg/ml), 10% Ino-RNA alone (3 µg/ml) or Ino-RNA incubated with increasing amounts of dsRNA-binding protein E3L at 50, 100 and 200 ng. Panel E, PHBE cells were treated with 10% Ino-RNA alone, with Ino-RNA and E3L, Ino-RNA and Galpha-i protein (Gp) (as a negative control). After 2 hr of incubation, total RNA was collected and RT-PCR reaction was performed (n≥3, ± SEM, *p<0.05, **p<0.01).
Figure 6.
Ino-RNA internalization is through SR-A-mediated endocytosis.
Panel A, Involvement of scavenger receptors in uptake of 10% Ino-RNA was determined by using dextran sulfate or fetuin. PHBE cells were treated with 10 µg/ml Cy3-labeled Ino-RNA for 5 min, or were treated with 10 µg/ml dextran sulfate or fetuin immediately before addition of Ino-RNA. After 5 min, cells were washed, mounted, and were used in confocal microscopy. Panel B, PHBE cells were treated with dextran sulfate or fetuin immediately before treatment with 10% Ino-RNA. After 2 hr total RNA was extracted and used in real-time RT-PCR. Panel C, PHBE cells were treated with endosomal acidification inhibitor BFA (100 nM) for 45 min and then 10% Ino-RNA for 2 hr. The total RNA was extracted and used in real-time RT-PCR. Error bars indicate standard error of the mean (n = 3, ± SEM, **p<0.01).
Figure 7.
Ino-RNA-induced signaling pathways.
Panel A, For analysis of innate inflammatory signaling pathways, PHBE cells were treated with Ino-RNA at 10 µg/ml and at indicated time points, total cellular protein extracts were prepared by cell lysis using 1X SDS-PAGE buffer. Phospho-proteins were probed using specific antibodies, and were visualized by the enhanced chemiluminescence detection. Panel B, For the activation of MAPKs detection, PKR, TLR3, MDA5 and RIG-I were treated by chemically synthesized siRNAs and then by 10% Ino-RNA. We next performed analysis of Ino-RNA induction of inflammatory cytokines following PKR, TLR3, MDA5 and RIG-I siRNA knockdown (Panel C, n = 3). Mock siRNA-treated cells were used as a control. Panel D, PKR-deficient mice or wild-type controls (4 in each group, n = 3) were treated intratracheally with 25 µl (1 mg/ml) of Ino-RNA or with 25 µl of PBS (vehicle control). After 4 hr, total lung RNA was isolated for RT-PCR (n = 3, ±SEM, *p<0.05, ** p<0.01).
Figure 8.
Global transcriptomic analysis.
Panel A, Ingenuity Bio-Function Analysis of genes upregulated (>2 fold) in 10% Ino-RNA-treated cells but not in normal RNA-treated cells. Panel B, Ingenuity Canonical Pathway Analysis of genes upregulated (>2 fold) in Ino-RNA treated cells but not in normal-RNA treated cells.
Table 1.
Microarray analysis of RNA from PHBE cells treated with Ino-RNA.
Table 2.
List of significant genes associated with antiviral and innate immune responses that are induced by 10% Ino-RNA and not by N-RNA.
Figure 9.
Proposed model of extracellular Ino-RNA-induced antiviral activity.