Regulatingimmuneresponsestoviralinfection

DonnaMacDuff,PhD

AssistantProfessorDepartmentofMicrobiologyandImmunology

dmacduff@uic.edu

MicrobeGrowth

UncontrolledReplication ImmunePathology

PathogenClearance

Howistheimmunesystemregulated?

MolecularmechanismsPhysiologicalrelevance

AnimalmodelsKnock-outmice

CellculturesystemsBiochemicalapproaches

Bacteria Viruses

CommonExperimentalApproachestoInterrogatetheImmuneSystem

Boisson etal.2012

WhyareHOIL1andLUBACsoimportantfortheimmunesystemtofunctionproperly?

HOIPattaches“linear”ubiquitinchainstootherproteinsHOIL1isalsoaubiquitinligase,butitsfunctionisunknown

Potentialfunctionsforubiquitinchainsattachedtoproteins:Facilitateproteininteractions/actasascaffoldMarkproteinsfordegradationbytheproteasomeActivateorinhibitproteinfunction

LUBAC=LinearUBiquitin chainAssemblyComplex

Linearubiquitinchainassemblycomplex(LUBAC)

Toll-likereceptorsInflammatorycytokines

Necroptosis RIG-I-likereceptorsInterferons

NOD-likereceptorsInflammasomePyroptosis

RodgersMAetal.,JEM2014InnKSetal.,MolCell.2011KhanMetal.,PLoSPathog.2016ZhangMetal.,CellResearch2008BelgnaouiSMetal.,CHM2012

ChattopadhyaySetal.,Immunity2016LiuSetal.,eLIFE2013

TokunagaFetal.,NatCellBiol.2009HaasTLetal.,MolCell.2009DamgaardRBetal.,MolCell2012HostagerBS,etal.,PloSOne2010ZakDEEtal.,PNAS2011others

ImmunefunctionsofLUBACandlinearubiquitination

LUBACregulatestheimmuneresponseaftersensingofpathogens

HOIL-1-/-

Highlysusceptibleto: Moreresistantto:

MacDuffDAetal.,eLIFE 2015

WhatarethephysiologicalfunctionsofLUBAC?HOIL-1-deficientmice– modelforhumandisease

Hyper-inflammatory?Intestinalabnormalities?

Immunodeficient?Cardiomyopathy?Murinenorovirus

Listeriamonocytogenes

Toxoplasmagondii

Mycobacteriumtuberculosis

Murinegamma-herpesvirus68

Citrobacter rodentium

MacDuffDAetal.,JVI2018

Currentstudiesinthelab

Commensalmicrobes

Type2inflammationintheintestine

Citrobacter rodentium(modelforE.coli)

SurvivalIntestinalbarrierintegrity

Murinenorovirus

TypeIandIIIInterferons

MDA5(notRIG-I)MacDuffetal.,JVI2018

HowdoesHOIL1balanceinflammationandinnateimmunitytocommensalandpathogenicmicrobesintheintestine?

On-goingstudiesinthelab

§ Closelyrelatedtohumannorovirusesthatcauseacutegastroenteritis

§ Endemicinmany(most)mousefacilitiesworld-wide

§ (+)ssRNA,non-envelopedvirus(Caliciviridae)

§ Infectslaboratorymiceinvivobygastro-intestinalinfection

§ Replicatesinmacrophagesanddendriticcellsinvitro

§ Interferonsareimportantforcontrollingacuteandpersistentinfection

Murinenorovirus(MNoV)

MurinenorovirusreplicatestohigherlevelsinHOIL1-deficientmice

10 20 302

3

4

5

6

7

Days after MNoV infection

Log 10

MN

oV c

opie

s/fe

cal p

elle

t

Hoil-/-Hoil+/+

WT Hoil1-/- WT Hoil1-/- WT Hoil1-/-

2

4

6

8

Log 10

MN

oV c

opie

s/ug

RN

A(n

orm

aliz

ed to

Rps29

)

Ileum Colon MLN

**** *

Viralsheddinginstool Viralburdensintissues

14dayspost-infection

B6

Hoil1-/-

0

1

2

3

4

IFNb transcript induction9 hpi MNV CR6 MOI = 3

log1

0 IF

Nb

fold

indu

ctio

n re

lativ

e to

WT

unin

fect

ed

HOIL1-deficientbonemarrow-deriveddendriticcellsmake100Xlessinterferon-betamRNAafterinfectionwith murinenoroviruscomparedtowild-typecells

~100x

IFN-bmRNA

HOIL1isimportantforinducinginterferonsduringmurinenorovirusinfection

WTHOIL1-/-

Howarevirusessensedbycellstoinduceinterferons?

Interferonsmadebyinfectedcellsactivateneighboringcellstoarmthemselvesagainstinfection.TheviralRNAsensor,MDA5,isimportantfortheinterferonresponsetoMNoV:“MDA-5recognitionofamurinenorovirus.”McCartneySAetal.,PLoS Pathog.2008

HallerOetal.,Virology2006

MNoVSeV

SeV (0

.1X)

VSV0

1

2

3

4

5

log1

0 IF

Nb

fold

indu

ctio

n re

lativ

e to

WT

untr

eate

d ControlHoil1-/-Mda5-/-

MNoVTMEV

poly(I:C

)

poly(I:C

) txn

0

1

2

3

4

5

log1

0 IF

Nb

fold

indu

ctio

n re

lativ

e to

WT

moc

k

ControlHoil1-/-Mda5-/-

IFN-b******

********

**

******

Bonemarrow-deriveddendriticcells

HOIL1isrequiredduringinfectionwithvirusessensedbyMDA5butnotwithvirusessensedbyRIG-I

10-100-folddefectsinIFNb mRNAinductionafterMNoV,TMEVinfectionorpoly(I:C)RNAtransfection– detectedbyMDA5.NodefectsinHOIL1-deficientorMDA5-deficientcellsafterinfectionwithSeV orVSV-detectedbyRIG-I.

MNoV =murinenorovirusSeV =SendaivirusVSV=vesicularstomatitisvirusTMEV=Theiler’smurineencephalomyelitisviruspoly(I:C)=syntheticdouble-strandedRNA

RNA

Interferonsa,b,l

ligand

receptor

adaptor

kinaseregulator

TranscriptionFactor

Transcript(mRNA)

SeV,VSVRNA

MAVS

IRF3/7

MDA5

TBK1/IKKeTANK/NEMO

HOIL1/LUBAC?

MNoV,TMEV

RIG-I?

HOIL1isrequiredforinductionofinterferonsdownstreamofMDA5,butnotRIG-I.ButMDA5andRIG-Iuseacommonsignalingpathway

HowdoesHOIL1regulatethesesignalingpathwaystocontrolviralinfection?

HOIL1/LUBAC?

• IdentifyproteindomainsofHOIL1(andHOIP,Sharpin)thatareimportantforinterferonproduction(generateandtestmutantgenes)

• IdentifyHOIL1-interactingproteins(testcandidateinteractors,developasystemtoidentifynewinteractors)

• Identifyproteinsthatareubiquitinated byHOIL1(testcandidateproteins,developasystemtoidentifyubiquitinated proteins)

• TestwhetherHOIL1activityalterspost-translationalmodifications(phosphorylation,ubiquitination,etc)onMDA5

• TestwhetherHOIL1expressionisimportantinmacrophages,dendriticcellsorintestinalepithelialcellsduringviralinfectioninvivo(infectmutantmiceandmeasureviralburdens)

PotentialRotationProjects

Questions? dmacduff@uic.edu

Labwebsite: https://sites.google.com/uic.edu/macduff/home