RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all

1. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription Qing Ge, Michael T. McManus, Tam Nguyen, Ching-Hung Shen, Phillip A. Sharp, Herman N. Eisen, and Jianzhu Chen Contributed December 23, 2002 Published March 4, 2003 Presented by Kelly McCoy

2. Why bother using RNAi to inhibit influenza production? 10-20% of the U.S. population is infected each year resulting in 40,000 deaths Influenza is easily spread Antigenic drift (changes in HA and NA- viral antigens) prevents immunity Antigenic shift (mixing of viruses from 2 different species) creates new strains that also prevent immunity

3. Brief Review of Influenza A 8 segments of ssRNA- Segments 1-6 each encode 1 protein, 7 & 8 each encode 2 proteins PB2, PB1, and PA are components of the RNA transcriptase HA, NA, and NP are the major glycoproteins Segment 7 encodes M1 and M2 and Segment 8 encodes NS1 and NS2

4. Brief Review of Influenza A Hemagglutinin (HA) is a viral protein anchored in the lipid bilayer that recognizes and binds to host cell�s sialic acid

5. Designing siRNAs specific for Influenza A virus Remember: RNAi uses dsRNA to direct sequence- specific degradation of mRNA Search for conserved sequences of 21 nucleotides (any longer sequence would trigger an interferon response) There are 15 HA subtypes and 9 NA subtypes, none of which have 21 n�tide conserved sequences Thus, Ge et al. designed 20 different siRNAs specific for NP, PA, PB1, PB2, M, and NS genes and tested them

6. Testing for Influenza Virus Production 2 testing methods were used: Inhibition of Flu production in cell lines Madin-Darby canine kidney (MDCK) cells were used siRNAs were introduced into the cell and then either A/PR/8/34 (PR8) virus WSN/33 (WSN) virus Inhibition of Flu production in Embryonated Chicken Eggs 10-day-old embryonated chicken eggs were used PR8 virus was introduced alone or in conjunction with the siRNAs

7. Inhibition of Influenza Production in Cell Lines Through electroporation, the 20 different siRNAs were introduced into the MDCK cells. 8 hours later, PR8 or WSN virus was added to the cell with a multiplicity of infection (moi) of 0.001, 0.01, or 0.1. Controls: 1. GFP-949 (siRNA specific for GFP) was introduced into MDCK cells expressing GFP. Later the cells were infected by the viruses. 2. Mock transfection: The virus was introduced into cells with no siRNA Using an HA assay, the virus titer was determined at different times after the infection for the controls and the tested cells

8. Results of siRNAs in Cell Lines infected with Influenza Figure A graphically represents the time vs. the virus amount for both PR8 and WSN virus Mock transfection virus titers increased over time GFP-949 did not affect virus production � this means that siRNA does NOT interfere nonspecifically with flu virus production

9. Results of siRNAs in Cell Lines infected with Influenza Together, Figures A and D show 3 different types of results Approx. 45% of the siRNAs had no effect on the virus titer Approx. 40% of the siRNAs significantly inhibited virus production Approx. 15% of the siRNAs potently inhibited production ***NP-1496 and PA-2087 produced no detectable HA activity

10. Results of siRNAs in Cell Lines infected with Influenza Figure B shows the potentcy of of siRNA- the virus titer was determined when MDCK cells were transfected with different concentrations of NP-1496 siRNA As the amount of siRNA decreased, virus titer increased but still very potent

11. Results of siRNAs in Cell Lines infected with Influenza Figure C shows that the procedure also works when reversed First the MDCK cells were infected by the virus and then by the siRNA Virus titer increased steadily with mock transfection but NP-1496 siRNA worked to keep the virus titer levels low

12. Summary of Cell Line Results Certain siRNAs potently inhibit flu production in MDCK cells Influenza production is inhibited by siRNAs specific for different viral genes (notably NP, PA, and PB1) siRNA works in cells with ongoing infection

13. Testing for Influenza Virus Production 2 testing methods were used: Inhibition of Flu production in cell lines Madin-Darby canine kidney (MDCK) cells were used siRNAs were introduced into the cell and then either A/PR/8/34 (PR8) virus WSN/33 (WSN) virus Inhibition of Flu production in Embryonated Chicken Eggs 10-day-old embryonated chicken eggs were used PR8 virus was introduced alone or in conjunction with the siRNAs

14. Inhibition of Influenza Production in Embryonated Chicken Eggs PR8 virus was injected into 10-day-old embryonated chicken eggs either alone or in conjunction with siRNA 17 hours later, virus titers were measured The controls used were the same as the ones testing the Cell lines- mock transfection (no siRNA) and GFP-949

15. Effects of siRNAs on Virus titer Mock transfection and GFP-949 had no effect on virus production The same siRNAs that potently inhibited virus production in MDCK cells (PB1-2257, PA-2087, NP-1496) considerably reduced virus titers in chicken embryos

16. What is most likely the direct target of RNAi? Figure A illustrates Influenza�s negative polarity The vRNA is copied into both cRNA (for replication purposes) and mRNA (to synthesize viral proteins) Thus what is the target of siRNA�mRNA, vRNA, or cRNA?

17. What is most likely the direct target of RNAi? Figure B show the effects on inhibition of virus production by modifying either the sense or antisense strands of the siRNA -Antisense siRNA strand is complementary to mRNA and cRNA -Sense siRNA strand is complementary to vRNA ***Inhibition requires a wt antisense strand in the siRNA- thus the target is either mRNA, cRNA or both

18. What is most likely the direct target of RNAi? Figure C distinguishes between the 3 RNAs MDCK cells were transfected with M-37 siRNA and PR8 RNA was isolated 1,2, and 3 hours later and quantified by RT (using primer shown in fig. A) and real-time PCR Gamma �actin was used to normalize the data ***The 50% reduction in mRNA in the presence of siRNA indicates that mRNA is the target

19. Some siRNAs Inhibit Accumulation of All Viral RNAs The same procedure was performed except that NP-1496 siRNA was used rather than M-37 siRNA

20. Some siRNAs Inhibit Accumulation of All Viral RNAs Figure A shows that 3 hours after infection, NP mRNA was detected only in the absence of NP-1496 But, NP-specific vRNA and cRNA were also inhibited in the presence of NP-1496 Figures B and C show that NP-specific siRNA inhibits the accumulation of M- and NS- specific mRNA, vRNA, and cRNA ***Therefore, depending on their sequence and specificity, some siRNAs have a global affect

21. Some siRNAs Inhibit Accumulation of All Viral RNAs There are 2 possible causes for this global inhibition of viral RNAs An interferon response is triggered by the presence of dsRNA -The same experiments were carried out in Vero cells (cells with the entire interferon locus deleted) and the results are shown in figure D -These graphs show that the response is the same with or without interferon present� PLUS�

22. Some siRNAs Inhibit Accumulation of All Viral RNAs The levels of transcripts from cellular genes was also assayed in the presence of siRNA The figure shows the level of NP (high after 3 hours with no NP-1496) compared to a ribosomal L32 gene (no difference is detected in the presence or absence of siRNA) Therefore, the broad inhibition of viral RNA is not due to a cellular interferon response

23. Some siRNAs Inhibit Accumulation of All Viral RNAs 2. The inhibition of viral RNA is a result of enhanced RNA degradation -The presence of dsRNA triggers a pathway that targets RNA for degradation. A major component of this pathway, PKR, was assayed first for cells with NP-1496 and no virus infection- no effect . Then PKR was assayed in cells infected with the virus. Virus levels increased both with and without NP-1496 -Therefore the increased level of RNA degradation is not the reason for the RNA inhibition

24. Summary of Important Findings siRNAs potently inhibit influenza virus production in both cell lines and embryonated chicken eggs siRNAs that target NP and PA are the most effective Some siRNAs exert their inhibitory effect by interfering with the accumulation of mRNA AND other viral RNAs (I.e. NP and PA are required for virus replication and translation) Viral mRNA is the direct target of siRNA-mediated interference

25. Implications The result of these analyses provide the beginning of exploration of siRNAs� possible role in an eventual therapy to treat influenza Epithelial cells of the respiratory tract are affected by the virus and therefore siRNAs could possibly be administered intranasally or pulmonarally

26. Sources Ge, Qing et al. �RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription� PNAS March 4, 2003 vol. 100 no. 5 p. 2718-2723 http://www.psc.edu/science/Herlocher/Herlocher.html http://microvet.arizona.edu/Courses/MIC419web/Case4flu.html Levine, Arnold J. Viruses 1992, New York p. 155-171