Paper presentation Lucas Kemper 06.12.2010

1. Paper presentation Lucas Kemper 06.12.2010

2. Introduction microRNA transport of pre-miRNA by Exportin 5 cleavage by Drosha in the nucleus cleavage by Dicer in the cytoplasm formation of the miRNA-induced silencing complex inducible expression of pri-miRNA with hairpin structure miRISC binding to 5‘or 3‘ UTR of mRNA block of mRNA translation higher mRNA instability mRNA cleavage Lindsay, A.M.; Cell, 2008

3. Introduction KSHV • Kaposi‘s sarcoma-associated herpesvirus • also known as human herpesvirus 8 (HHA8) • structure • can induce several human cancers Kaposi‘s sarcoma LEC • primary lymphatic endothelial cells

4. Aim innate immune response uninfected cell infected cell microRNA antiviral state determine the role of host microRNAs in the innate immune response during virus infection

5. Effect of KSHV infection on the miRNA profile of LECs qRT-PCR microarray • several host miRNAs upregulated by KSHV infection • identification of two groups of upregulated miRNA (early / late)

6. Testing potential role of identified miRNAs in the antiviral response 2d after infection • inhibition of two early miR suppresses viral gene expression • inhibiting late miR has no effect on viral gene expression

7. How to continue? miR-132 vs. miR-146a • Implication in neuronal differentiation and function • Induced after LPS-stimulation of monocytic cell lines • Overexpressed in chronic lymphoblastic leukaemia • regulated by CREB miR-132 primary miR transcript less stable weaker induction lower effect miR-212

8. miR-132 induction is a result of viral binding and not of viral gene expression fold increase to uninfected • miR-132 induction is fast an transient • miR-132 induction based on viral binding

9. miR-132 is induced through a CREB-dependent mechanism kinetics of CREB phosphorylation chemical inhibition of MAPKs siRNA knock down of CREB & ERK1/2 CREB phosphorylation dependent on MAPKs • induction of miR-132 mediated by ERK-CREB dependent pathway

10. p300 is a target of miR-132 • Using bioinformatics: • PITA • TargetScan • EiMMO • Miranda Search algorithms to predict binding between microRNAs and mRNAs p300 is a putative target of miR-132  co-activator  interacts with CREB  essential for initiation of antiviral immunity  miR-132 targets an 8mer site in the UTR of p300

11. p300 is a target of miR-132 transfected contructs transfected contructs LECs LECs • miR-132 reduces p300 protein level which is functionally relevant

12. p300 is a target of miR-132 6h p.i. fold increase to uninfected • miR-132 induces its own feedback loop • feedback loop explains partly transient miR-132 induction

13. miR-132 inhibits the antiviral interferon response and enhances viral replication • inhibition of miR-132 leads to • more IFN-β and ISG expression • less KSHV replication

14. miR-132 regulates viral gene expression through suppression of p300 KSHV • p300 protein level is reduced • due to miR-132 • viral mRNA level is increased • due to miR-132

15. Summary

16. Summary This work reveals: a viral gene expression-independent, host-microRNA-mediated mechanism that regulates the antiviral response.

17. Thank you

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19. wt or mutated miR132_212 cluster + Renilla UTR HeLa • miR-132_212 cluster represses a construct • containing p300 UTR

20. miR-132 induced repression of p300 • affects p300 regulated gene expression

21. miR-132 regulates viral gene expression through suppression of p300 viral genes • knock down of p300 reduces antiviral resonse after KSHV infection • knock down of p300 promotes viral gene expression