Nature , 2008, Doi:10.1038/nature07103

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

Background Information • HSV-1 • miRNA

envelope capsid tegument & capsid associated proteins TRL UL IRL IRS US TRS 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 U: unique sequence S: short R: repeat sequence T: terminal L: long I: inverted

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

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 Gottwein & Cullen, 2008 UTR: untranslated region

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

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 miRNA formation & processing www.ambion.com

pre-miRNAs transported to cytoplasm via an exportin 5-dependent mechanism 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 miRNA formation & processing RISC: RNA-induced silencing complex RNAi: RNA interference

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 miRNA formation & processing

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

HSV-1 Research Paper Specific Results

Genomic location of HSV-1 miRNAs

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

Predicted secondary structures of HSV-1 miRNA precursors

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

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

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

Summary

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

Thank You For Your Attention!

Nature , 2008, Doi:10.1038/nature07103