1. Nature, 2008, Doi:10.1038/nature07103 Semrah Kati 09.02.2009

2. Background Information HSV-1HSV-1 miRNAmiRNA

3. Herpes Simplex Virus-1 (HSV-1) family: herpesviridae subfamily: α-herpesviridae latent & lytic life cyle diameter of 120-200nm enveloped genome: linear dsDNA, 152 kbp with over 80 ORFs envelope capsid tegument & capsid associated proteins TR L U L IR L IR S U S TR S U: unique sequence S: short R: repeat sequence T: terminal L: longI: inverted

4. HSV-1 life cycle & infection routes Frampton et al., 2005

5. microRNAs (miRNAs) small, non-coding, single-stranded RNA molecules 19-23 nucleotides imperfect stem-loop structures encoded by plants, animals and viruses mammalian genomes are predicted to encode 200-500 miRNAs regulate gene expression by binding to the 3´UTR of specific mRNAS (translational repression) processed by 2 ribonuclease-III enzymes UTR: untranslated region Gottwein & Cullen, 2008

6. miRNA formation & processing Biogenesis & post transcriptional suppression of miRNAs and siRNAs. He & Hannon, 2004 overview

7. miRNA formation & processing miRNAs initially transcribed as part of a long RNA molecule Primary-miRNA (500-3000nt) in the nucleus Drosha (dsRNA-specific ribonuclease) processes pri-miRNAs into 70-100nt hairpins RNAs Pre-miRNA Drosha acts in conjunction with RNA- binding protein DGCR8 www.ambion.com

8. miRNA formation & processing pre-miRNAs transported to cytoplasm via an exportin 5-dependent mechanism RISC: RNA-induced silencing complex RNAi: RNA interference in cytoplasm the pre-miRNAs are digested by a second ds-specific ribonuclease (Dicer) resulting in a sequence-specific, ss-, mature miRNA molecule ss-, 19-23nt mature miRNA bound by a complex similar to RISC that participates in RNAi

9. miRNA formation & processing RISCs consist minimally of the miRNA and one of four Argonaute (Ago) proteins in animals, the RISC-bound miRNA binds mRNAs through a sequence that at least partially matches the 3`-UTR of target mRNA Seed region: 2-7 nt from the miRNA 5`end bound mRNA remains untranslated, resulting in reduced expression of the corresponding gene

10. Viral miRNA… exclusively derive from dsDNA viruses mainly of the herpesvirus family e.g: HSV-1, KSHV, EBV, hCMV regulate viral and/or host cell gene expression may regulate viral replication and pathogenesis

11. Paper Specific Results HSV-1 Research

12. Genomic location of HSV-1 miRNAs

13. LAT expression after pcDNA3-LAT transfection into 293T cells Nothern Blot:

14. Predicted secondary structures of HSV-1 miRNA precursors

15. HSV-1 pre-miRNAs quantitative RT-PCR: 293T transf. with pcDNA3-LAT Vero inf. with HSV-1 mouse trigeminal ganglia latently inf. with HSV-1

16. Downregulation of ICP0 protein expression by HSV-1 miR-H2-3p WB of ICP0: Co-transfection of β–Arrestin, ICP0 WT or mutant & miR-H2-3p or miR-H2-3p-3M Nothern Blot:

17. Downregulation of ICP4 protein expression by HSV-1 miR-H6

18. Summary

19. LAT is a primary miRNA precursor & encodes 4 distinct miRNAS. miRNA-H2-3p is transcribed in antisense orientation to ICP0 & reduces ICP0 protein expression at the translational level. Discovery of the 5th miRNA, miR-H6, deriving from a previously unknown transcript distinct from LAT. miR-H6 reduces ICP4 protein expression. ability of 2 primary miRNA precursors in latently infected neurons to promote the establishment & maintenance of viral latency by post-transcriptionally regulating viral gene expression

20. Thank You For Your Attention! Your Attention!