5'PPP-RNA suprresses the replication 1918 virus as well as wild-type and drug-resistant H5N1 viruses. (A) A549 (1 × 10⁶ cells/well) in a 6-well tissue culture plate were mock-transfected (control) or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA using lipofectamine 2000 and 24 hr later were infected with (i) 1918 pandemic virus (0.01 MOI). (ii), Transfected A549 cells were also infected with wild-type H5N1 (0.1 MOI) following 0, 24 and 48 hr of transfection. B. Mock, 5'PPP-RNA or CIAP-RNA transfected A549 cells were infected with drug-resistant H5N1 viruses A/VN/1203/2004 (i), A/VN/30408/2004 H274Y(ii) and A/VN/30408/2004 N294S (iii) 24 hr post transfection. Supernatants collected after 24 hr of infection were assayed for viral titers and results shown are mean ± SD of three independent experiments and are expressed as viral titer (pfu/ml).

5'PPP-RNA induced RIG-I and IFNβ expression in A549 cells. (A) A549 (1 × 10⁶ cells/well) in a 6-well tissue culture plate were mock-transfected (control) or transfected with 2 μg of 5'PPP-RNA, CIAP-RNA or chemically synthesized OH-RNA using lipofectamine 2000. After 24 hr of treatment, (i) IFNβ and (ii) RIG-I mRNA expression was analyzed by real-time RT-PCR. All data were normalized to β-actin, a house keeping gene and expressed as fold increase. Data shown represent the mean ± SD of three independent experiments. (B), (i) A549 cells were transfected with 2 μg of 5'PPP-RNA for the indicated times, and RIG-I expression was analyzed by immunoblot. (ii) A549 cells were mock-transfected or transfected with 2 μg of 5'PPP-RNA, CIAP-RNA or chemically synthesized OH-RNA for 24 hr. RIG-I expression was analyzed by immunoblot. (iii) A549 cells grown on cover-slips were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr. Cells were then paraformaldehyde fixed and immunostained with anti-RIG-I antibodies. Alexa fluor 594 goat anti-rabbit IgG Antibody (red fluorescence) or Alexa 488 (green fluorescence) were used as secondary antibodies. Nuclei were stained with Hoechst (blue fluorescence). (C) RNA was isolated from A549 cells mock transfected or trasfected with 5'PPP-RNA or CIAP-RNA for 24 hr and then infected with wild-type H5N1 virus for 24 hr (as described in Figure legend 1). Real-time RT-PCR was performed to analyze the expression of (i) IFNβ, (ii) RIG-I and (iii) NS1. All data were normalized to β-actin, a house keeping gene and expressed as fold increase. Data shown represent the mean ± SD of three independent experiments.

Therapeutic potential of 5'PPP-RNA. A459 cells (1 × 10⁶ cells/well) in a 6-well tissue culture plate were infected with (i) wild-type A/New York/02/2001 and its (ii) H274Y oseltamivir-resistant A/New York/02/2001 variant for the 0 hr, 4 hr or 8 hr before treatment with 5'PPP-RNA or CIAP-RNA. Supernatants collected after 24 hr of infection were assayed for viral titers as indicated in material and methods. Cells were harvested to assay IFNβ (iii) and RIG-I (iv) mRNA level by Real time RT-PCR. Results shown are mean ± SD from three independent experiments and are expressed as viral titer (pfu/ml) or fold increase over controls.

5'PPP-RNA inhibits replication of 2009 pandemic influenza virus. (A). A459 cells (1 × 10⁶ cells/well) in a 6-well tissue culture plate were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr and then infected with A/California/08/09 at an MOI of 1.0. Supernatants collected 24 hr post-infection were assayed for viral titers as indicated in material and methods. Results shown are mean ± SD from three independent experiments and are expressed as viral titer (pfu/ml) (B). 5'PPP-RNA also reduced replication of A/Mexico/4482/09 virus in Balb/c mice. 8 week old female Balb/c mice received 100 μg of 5'PPP-RNA or PBS alone intravenously for 4 days and the animals were infected with 1000 MID₅₀ of 2009 pandemic virus (A/Mexico/4482/09) on day 1. The lungs were collected on day 4 to determine viral titers *P < 0.05 (n = 5).

Indispensible role of type I interferon in RIG-I-dependent 5'PPP-RNA-induced antiviral effects. (A). A549 cells (1 × 10⁶/well) in a 6-well plate were transfected with control siRNA or siRNA against RIG-I or IFN αβ receptors using DharmaFect 1 transfection reagent as per manufacturer instructions (Dharmacon, Lafayette, CO, USA). After 24 hr, cells were transfected with 2 μg of 5'PPP-RNA. A549 cells were then infected with A/New York/02/2001, a H1N1 virus at 0.1 MOI. Supernatants collected after 24 hr were analyzed for virus growth by plaque assay using MDCK cells. (B) A549 cells from experiment (A) were also analyzed for IFNβ and RIG-I expression by quantitative RT-PCR as described elsewhere. Data shown represent the mean ± SD of three independent experiments.