Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 May 21:7:102.
doi: 10.1186/1743-422X-7-102.

5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

Priya Ranjan  1 , Lakshmi Jayashankar, Varough Deyde, Hui Zeng, William G Davis, Melissa B Pearce, John B Bowzard, Mary A Hoelscher, Victoria Jeisy-Scott, Mayim E Wiens, Shivaprakash Gangappa, Larisa Gubareva, Adolfo García-Sastre, Jacqueline M Katz, Terrence M Tumpey, Takashi Fujita, Suryaprakash Sambhara
Affiliations

5'PPP-RNA induced RIG-I activation inhibits drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza virus replication

Priya Ranjan et al. Virol J. .

Abstract

Background: Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.

Results: In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication in vivo in mice.

Conclusions: Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status.

PubMed Disclaimer

Figures

Figure 1
Figure 1
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).
Figure 2
Figure 2
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).
Figure 3
Figure 3
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.
Figure 4
Figure 4
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.
Figure 5
Figure 5
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K. Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA. 2003;289:179–86. doi: 10.1001/jama.289.2.179. - DOI - PubMed
    1. Zambon MC. Epidemiology and pathogenesis of influenza. J Antimicrob Chemother. 1999;44(Suppl B):3–9. doi: 10.1093/jac/44.suppl_2.3. - DOI - PubMed
    1. Zambon MC. The pathogenesis of influenza in humans. Rev Med Virol. 2001;11:227–41. doi: 10.1002/rmv.319. - DOI - PubMed
    1. Nicholson KG, McNally T, Silverman M, Simons P, Zambon MC. Influenza-related hospitalizations among young children in Leicestershire. Pediatr Infect Dis J. 2003;22:S228–30. - PubMed
    1. OIE. Update on avian influenza in animals (type H5) 2008. http://www.oie.int/downld/AVIAN%20INFLUENZA/A_AI-Asia.htm Accessed on 02/29/2008.

Publication types

MeSH terms