Nature, 2008, Doi:10.1038/nature07103

Semrah Kati09.02.2009

Background InformationBackground Information

•HSV-1HSV-1•miRNAmiRNA

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

envelopeenvelope

capsidcapsidtegument & tegument & capsid associated capsid associated proteinsproteins

TRL UL IRL IRS US TRS

U: unique sequence S: shortR: repeat sequence T: terminalL: 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

UTR: untranslated region

Gottwein & Cullen, 2008

miRNA formation & processing

Biogenesis & post transcriptional suppression of miRNAs and siRNAs.He & Hannon, 2004

overview

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 RNAsPre-miRNADrosha acts in conjunction with RNA-binding protein DGCR8

www.ambion.com

miRNA formation & processing

pre-miRNAs transported to cytoplasm via an exportin 5-dependent mechanism

RISC: RNA-induced silencing complexRNAi: 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

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

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

Paper Specific ResultsPaper Specific Results

HSV-1 Research

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

HSV-1 pre-miRNAs

quantitative RT-PCR:

293T transf. with pcDNA3-LATVero inf. with HSV-1mouse trigeminal ganglia latently inf. with HSV-1

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:

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

SummarySummary

• 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

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