Vi C ll d DiViruses, Cells and DiseaseNovember 13, 2008

C i ( C )Human Cytomegalovirus (HCMV)Immediate early proteins, gene expression and signaling

QuickTime™ and a decompressor

are needed to see this picture.Dr. Hua ZhuICPH E350DUMDNJ - New Jersey Medical School973-972-4483 X 2-6488zhuhu@umdnj.edu

Electron Cryomicroscopy and 3D ReconstructionFrom Dr. H. Zhou

IntroductionIntroduction1. Medical aspects of HCMV1. Medical aspects of HCMV2. HCMV structure2. HCMV structure3. HCMV replication3. HCMV replication

Special topicsSpecial topics1. IE1 and IE2 functions1. IE1 and IE2 functions2. A HCMV2. A HCMV--induced signal transduction pathwayinduced signal transduction pathway

(di t t k)(di t t k)(discuss two papers next week)(discuss two papers next week)3. Functional profiling of HCMV genome3. Functional profiling of HCMV genome4. Identification of HCMV pathogenic genes4. Identification of HCMV pathogenic genesp g gp g g

Virus• Herpesvirus, dsDNA virus• ~80% of adults are infected• ~80% of adults are infected

Transmission• in utero and at any age

- body fluids• breast feeding, blood transfusion

Latency and Recurrence• Latent in monocytes (Myeloid progenitor cells)• Reactivates during pregnancy and in

immunosuppressed states, resulting in cytomegalic inclusion disease (CID), CMV pneumonia retinitis and hepatitisCMV pneumonia, retinitis and hepatitis

Pathogenicity• In people with cancer, transplanted organs, AIDS or other

immune deficiencies CMV can cause severe diseases of theimmune deficiencies, CMV can cause severe diseases of the lung, liver, colon, eye or brain

• A leading cause of congenital birth defects

HCMV infection in the lung

Intranuclear inclusion with a clear zone

Transplant RecipientsTransplant RecipientsImmunosuppression

R t f CMV I f ti d di i li t l t

Pretransplant

Rates of CMV Infection and disease in liver transplant patients according to types of infection

Pretransplant serostatus of

donor/recipientType of infection Infection

(%)Disease

(%)+/+ Reinfection or 66 23+/+ Reinfection or

reactivation66 23

+/- Primary 77 61

-/+ Reactivation 49 10

-/- Primary, not from graft 10 10

HCMV and AIDSC i di t f d th i AIDS (25%)• Common immediate cause of death in AIDS (25%)

• 70-90% show active CMV infection• 30-40% develop CMV retinitis• 95% homosexual men with AIDS have active HCMV infection

CMV retinitis

Congenital HCMV Infection

The most common virus globally to be transmitted in utero,affecting up to 2.5% of all live births (WHO) A leading cause of birth defectsA leading cause of birth defects~10% of infected fetuses exhibit damage of the CNS-mental retardationhearing losshearing lossimpaired vision

Prevention and treatment

• Vaccines: AD169, Towne - not effective

• Antivirals: Ganciclovir - effective

Anti Herpesvirus AgentsAnti-Herpesvirus Agents QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Acyclovir (Zovirax - Glaxo) - for α herpesviruses Ganciclovir (Cytovene - Roche) - for β herpesviruses

- Guanosine analogs

Mechanism of Action:• Specifically activated by virus-induced kinases,

such as thymidine kinase of HSV and UL97 of CMV• Terminate viral DNA chain elongationTerminate viral DNA chain elongation• Selectively inactivate viral DNA polymerases and inhibit

viral DNA elongation

Acyclovir

Viral kinase

C ll l ki

Acyclovir monophosphate

OH

Ganciclovir Cellular kinase

Viral DNA l

Acyclovir triphosphate

polymerase

Chain termination

HCMV Virion Structure

l

icosahedralnucleocapsiddsDNA genomeenvelopeglycoprotein tegument

H. Zhu

HCMV Genomic Structure

CMV (230kb)CMV (230kb)

AD169T

UL US

TowneToledoPH

20 40 60 80 100 120 140 160 180 200 220 230 kb0

TRFIX

HCMV Encodes 230-250 Potential ORFs

Murphy et al, 2003

Early Events of HCMV Infection

HSPG

HCMV

gB

gM/N

gHgM/N

HSPGgBgH

EGFR?Integrins

IE

Overview of HCMV Gene Expression Patterns

Cycloheximide

IE IEcTFs ?

IE E E EL

IEcTFsvTPs X X X X

X

IE E E EL

UL122 (IE2), UL123 (IE1),UL122 (IE2), UL123 (IE1),UL36, UL37, US3, UL119, TRS1 (IRS1)

HCMV Major IE Locus

Major IE promoter

E1 E2 E3 E4 E5

Major IE promoter

TATA

E1 E2 E3 E4 E5IE1 mRNAIE1 protein (72 kD)

ATF/CREBAP1SP1NF 1

IE2 mRNAIE2 protein (86 kD)

NF-1NF-κB

Is IE2 essential for HCMV replication? Is IE2 essential for HCMV replication?

Construction of HCMVConstruction of HCMV--BACBAC

HCMV DNA ProduceHCMV DNABAC vector

Transfect HF cells

Produce infectious HCMV

(GFP + CmR)

Homology recombination

Transfect HF cells

P if HCMVMutagenesis• Specific • Random

HCMV-BAC

Purify HCMV-BAC DNA

Purify recombinant

• Transform into E. coli• Select CmR colonies• Screen full-length HCMV-BAC

yHCMV (Green)

• Infect new HF cells• Isolate circular viral DNA

g

Generation of Recombinant HCMV Generation of Recombinant HCMV

Select for kanR

colonies at 32°C. . .

. .ORF

Flanking

kanRPCR kanR cassetteTransform into DY380

kanR

Incubate at 42°C Preparing electro-competent cells

Confirm recombinant HCMV-BAC by PCR and Southern

gseqs. (40 bp)

ORFE. coli DY38032°C

exo bet gam cI

HCMV-BACDefective λ prophage

competent cells

ΔORF mutant

kanR

Isolate exo bet gam cI Degrades

processively from the 5’ ends of the break sites

Inhibits the recBCD exonuclease from

ts λ cI repressorBAC DNA

Transfect HF cellsbreak sites

Binds to the remaining 3’ single strand tail, protecting and

exonuclease from attacking linear DNA

Produce infectious

?

, p gpreparing the recessed DNA for homologous strand invasion

infectious HCMV

Dr. Neal Copeland

IE2 is essential for viral replicationIE2 is essential for viral replication

MIEP

TATA WTE1 E2 E3 E4 E5

IE2ΔDeletion of exon 5

X

WT

IE2Δ WT

IE2ΔX

anti-IE1Genotype Plaques

anti-IE2

yp q

WT 223

IE2Δ 0

IE2 is required for the early and late gene expression

I di t E l E l

IE2 is required for the early and late gene expression

W Δ W Δ W Δ W Δ W Δ

IE1 IE2 TRS1 TRL4 UL112

Immediate Early Early

1018

506/517506/517396

344298

220

Do IE1 and IE2 directly regulate viral gene expression?Do IE1 and IE2 directly regulate viral gene expression?

IE1 IE2 ? ? ?

IE E EL

ChIPChIP--Chip AssayChip Assay

TFs Viral promoter?IE1IE2

TFs

Formaldehyde treat cellsIsolate nuclei

SonicateSonicate

Anti-HCMV IE1/2 Immunoprecipitate (anti-IE1/2)

Reverse crosslinksIdentify target DNA

Anti-HCMV IE1/2or Anti-HSV gB

Identify target DNA

ChIPChIP--Chip AssayChip Assay

202 kb 204 kb 206 kb 208 kb 210 kb 212 kb 214 kb 216 kb 218 kb

US12 US14 US16 US18 US20 US22 US23 US24 US25 US26 US27 US28US13 US15 US17 US19 US21

202 kb 204 kb 206 kb 208 kb 210 kb 212 kb 214 kb 216 kb 218 kb

PCRPCR

HCMV genomic chipInput

IPInput + IP

Control (9-mer)

Anti-HSV gB Anti-HCMV IE1/2

Merge

IE2IE1 IE2

• IE2 is essential for viral replication

IE E EL

IE2 is essential for viral replication• IE2 is required for viral early and late gene expression• IE1 and IE2 directly regulate viral gene expression

What Are the Host Response to HCMV?What Are the Host Response to HCMV?

HCMV

gH

gM/N

HSPG

HCMVR

gBg

IEIE

?

Identification of cellular genes altered after HCMV infection

Differential displayDifferential displayGeneChip (Affymetrix)cigs (IFN-stimulated genes, isgs)Activated by both live and killed HCMVNot require viral gene expression, viral DNA,

and new protein synthesisand new protein synthesisNot mediated by IFNs or other cytokinesHCMV directly activates ISGs

cig34

cig6 cig24 cig25

cig41cig1 cig52 cig53

cig43cig42

cig49 control

cig1 = isg54k

ISREGASGAS

What is required for induction of ISGs?What is required for induction of ISGs?

HSPG

HCMV gB

gH

gM/N

HSPG

HCMVR

gH

??

IE

ISG

gM/N

Is fusion required?Is fusion required?

HSPG

HCMV gB

gH

gM/N

PEG

HCMVR

- W W M MHCMV: CFI

isg54K -

cig49 -

CFI: - + +--

7 SK -

HCMV: - + ++-

ISG ?isg54K -

CFI: - - ++-PEG: --

+-+HCMV: + ++

7 SK -

No growth: 45; Severe growth: 12; Moderate growth defect: 23; Growth like WT: 68; Enhanced growth: 4

Dunn et al., 2003, PNAS

Tissue Tropism Tissue Tropism

A: Fibroblast cells; B. Epithelial cells; C. Endothelial cells

CMV (240kb)CMV (240kb)

AD169T

UL US

TowneToledoPH

20 40 60 80 100 120 140 160 180 200 220 230 kb0

TRFIX

The Severe Combined Immunodeficient (SCID) Mice The Severe Combined Immunodeficient (SCID) Mice Model For Studying HCMV PathogenesisModel For Studying HCMV Pathogenesis

thymus/liver tissue

y g gy g g

kidney kidney capsule

3 months

/

Infected implants are harvested at different time

d th t f HCMV A thymus/liver implant is surgically exposed and injected with HCMV

and the amount of HCMV is measured

The clinical but not attenuated strains can replicate in The clinical but not attenuated strains can replicate in ppthe implanted human tissuesthe implanted human tissues

Fibroblasts SCID-huFibroblasts SCID hu

Attenuated strains:AD169, Towne

Yes No

Clinical strains:Toledo

Yes Yes

Construction of A HCMV with a Luciferase Gene

Luciferase (UL62/UL63)

Toledo

Luciferase (UL62/UL63)

Luciferase

30000000

Luciferase

15000000

20000000

25000000

0

5000000

10000000

0HCMVBAC HCMVluc

Bioluminescence Imaging Technique (Xenogen IVIS® Imaging System)(Xenogen IVIS® Imaging System)

A. Virus w/ Luc. B. Animal/cell models C. Image acquisition D. Data analysis

1 2 3 4 5 6 7 8

Toledo ToledoL cToledo ToledoLuc

Generation of ToledoGeneration of Toledo--D and ToledoD and Toledo--R MutantsR Mutants

AD169Towne

UL US

TowneToledoToledo-DToledo-R

Cultured cells SCID-hu (Thy/Liv)

AD169 Yes No

Toledo-R

AD169 Yes NoTowne Yes NoToledo Yes YesToledo-D Yes NoToledo-R Yes Yes

The 15-kb Segment Contains the Crucial Genes for HCMV Replication in vivo

AD169Towne

UL US

TowneToledoPHTR

20 40 60 80 100 120 140 160 180 200 220 230 kb0

TRFIX

UL133 UL135 UL136 UL139UL138

UL142UL141UL143

UL145 UL147 UL148 UL132 UL130 UL149UL133

UL134

UL135 UL136

UL137

UL139 UL142

UL140

UL141

UL144

UL145

UL146

UL147 UL148 UL132 UL130 UL149

UL150

UL151