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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