Paper presentation

Lucas Kemper

06.12.2010

Introduction

microRNA

Lindsay, A.M.; Cell, 2008

transport of pre-miRNAby Exportin 5

block of mRNA translationhigher mRNA instability

mRNA cleavage

inducible expressionof pri-miRNA withhairpin structure

cleavage by Droshain the nucleus

cleavage by Dicerin the cytoplasm

miRISC binding to5‘or 3‘ UTR of mRNA

formation of themiRNA-induced

silencing complex

Introduction

KSHV

Kaposi‘s sarcoma

Kaposi‘s sarcoma-associated herpesvirus

also known as human herpesvirus 8 (HHA8)

structure

can induce several human cancers

LEC

primary lymphatic endothelial cells

Aim

determine the role of host microRNAs in the innate immune response

during virus infection

innate immune response

uninfectedcell

infectedcell

antiviral state

microRNA

Effect of KSHV infection on the miRNA profile of LECs

microarrayqRT-PCR

several host miRNAs upregulated by KSHV infection identification of two groups of upregulated miRNA (early / late)

Testing potential role of identified miRNAs in the antiviral response

2d

aft

er

infe

ctio

n

inhibition of two early miR suppresses viral gene expression inhibiting late miR has no effect on viral gene expression

miR-132 miR-146a

How to continue?

vs.

Implication in neuronal differentiation and function

Induced after LPS-stimulation of monocytic cell lines

Overexpressed in chronic lymphoblastic leukaemia

primary miRtranscript

miR-132

miR-212 less stableweaker inductionlower effect

regulated by CREB

fold

incr

ease

to u

ninf

ecte

d

miR-132 induction is a result of viral bindingand not of viral gene expression

miR-132 induction is fast an transient

miR-132 induction based on viral binding

miR-132 is induced through a CREB-dependent mechanism

kinetics of CREBphosphorylation

chemical inhibitionof MAPKs

siRNA knock downof CREB & ERK1/2

CREB phosphorylationdependent on MAPKs

induction of miR-132 mediated by ERK-CREB dependent pathway

p300 is a target of miR-132

Using bioinformatics:

PITA TargetScan EiMMO Miranda

Search algorithms to predict bindingbetween microRNAs and mRNAs

p300 is a putative target of miR-132

miR-132 targets an 8mer site in the UTR of p300

co-activator

essential for initiation of antiviral immunity

interacts with CREB

p300 is a target of miR-132

LEC

s

transfected contructs

LEC

s

miR-132 reduces p300 protein level which is functionally relevant

transfected contructs

p300 is a target of miR-132

6h p.i.

fold

incr

ease

to u

ninf

ecte

d

miR-132 induces its own feedback loop

feedback loop explains partly transient miR-132 induction

miR-132 inhibits the antiviral interferon responseand enhances viral replication

inhibition of miR-132 leads to

less KSHV replication

more IFN-β and ISG expression

miR-132 regulates viral gene expressionthrough suppression of p300

p300 protein level is reduced due to miR-132

KSHV

viral mRNA level is increased due to miR-132

Summary

This work reveals:

a viral gene expression-independent, host-microRNA-mediated mechanism that regulates the antiviral response.

Summary

Thank you

…..

RenillaUTR

HeLa

+wt or mutated

miR132_212 cluster

miR-132_212 cluster represses a construct containing p300 UTR

miR-132 induced repression of p300 affects p300 regulated gene expression

miR-132 regulates viral gene expressionthrough suppression of p300

viral

gene

s

knock down of p300 reduces antiviral resonse after KSHV infection

knock down of p300 promotes viral gene expression