1. Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology, Chulalongkorn University Hospital. Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand. Department of Medicine, Chulalongkorn University Hospital.

2. SiRNA miRNA

3. BMJ 2004;328:1245–48

5. Targets for gene silencing Host proteins Leader RNA (Yang et al 1998,1999) Viral proteins N ( Wunner et al., 1991, Wu et al 2002) P (Morimoto et al. 2005, Blondel et al.,2006) L (Tordo et al., 1998) G M (Finke,Conzelmann 2003) Viral genome (Barik 2002)

6. wash Plating 70% conf 12 hours Plasmid siRNA virus transfection 12 hours infection 8 hours 24 hours 2 viral titers Cellular RNA Supernatant IF Western blot Cellular RNA Supernatant Western blot Real-time PCR

7. Pcmv 10-4 24 hr in cell We tested 5 anti-N 2 anti-leader and 3 anti negative strands

8. SiRNA PS17 CONTROL Control and transfected neuro-2a cells that express the PS17 siRNA were infected with CVS rabies virus for 48 h before they were fixed in acetone and stained with a FITC-labeled anti-N monoclonal antibodies

9. Conclusions from siRNA studies Specific viral mRNA and viral replication can be reduced by siRNA Inhibition of leader RNA and negative strand RNA is less effective than inhibiting N mRNA All methods that we tested (22 nt siRNA, PCR- based, vector with pol II or pol III promoter ) normally yield only 70% reduction of viral protein mRNA and viral genome Stable NA cell line expressing anti-N siRNA slow the growth rate of the virus

10. the siRNA is extremely stringent in its specificity such that a single nucleotide mismatch may abrogate its function To develop therapeutic tool- Need method that is more effective and less stringent

11. miRNA

12. Approximately 60%of miRNAs are expressed independently, 15% of miRNAs are expressed in clusters, and 25% are in introns.

13. miRNA - 24 hr in cell All anti-N miRNA tested can suppress 85- 95% of viral transcription Not significantly affect house keeper gene Can suppress 85- 98 % of viral genome production

14. 85 % to 99 % suppression of virus production as measured in supernate

15. Stained with anti G AbStained with anti N Ab miRNA- NP1 Control neuro-2a IF 48 hours after challenged with same amount of CVS virus

16. miRNA against G mRNA Viral genome in cells 24 hours after infection Relative amount of viral RNA

17. miRNA against viral genome Relative amount of viral RNA in neuro-2a 72 hours after infection

18. miRNA against G-mRNA and against negative strand viral genome are not effective

19. Target 1 Target 2 Target 3 Are 3 copies of same miRNA more effective? Is miRNA against 3 different targets more effective? When N protein is inhibited, is the viral genome more vulnerable to miRNA? Does incorporation of ineffective miRNA to the complex reduce its activities? 3 targets -> more chances of counter genetic variability and mutation

20. Effect of expressing more than one miRNA as a chain Viral RNA in supernatant 48 hours after infection Relative amount of viral RNA

21. Effect of expressing more than one miRNA as a chain Relative amount of RNA in cell 24 hours after infection

22. Effect of expressing effective miRNA together with ineffective miRNA Relative amount of viral RNA in neuro-2a cells 72 hours after challenged with CVS

23. Chaining miRNA against viral genome with miRNA against NmRNA Viral RNA in supernatant 72 hours after infection Relative amount of viral RNA

24. Can RNA interference be used for street rabies?

25. Effect of dsRNA and miRNA (designed against CVS virus) on street virus NmRNA in neuro-2a cells 24 hours after infection 85 % reduction of viral transcription

26. HEP-Flury –GFP (from Dr. Morimoto) controldsRNA miRNA-N Still reasonably effective against targets that have 2-3 nucleotides different from designed targets

27. Conclusions miRNA against N-mRNA is effective in reducing both transcription and replication miRNA against viral genome alone is not effective Expressing multiple miRNA as a single premiRNA transcript is possible and will not significantly reduce effectiveness of individual miRNA

28. Conclusions miRNA can inhibit (at least) some strains of virus that contain different nucleotide(s) at miRNA target site

29. Acknowledgements T. HEMACHUDHA N. RATANASETYUTH P.SUPAVONWONG P. VIROJANAPIROM P. KHAWPLOD BIOTEC Thailand