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 [email protected]
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 Genomic Structure
CMV (230kb)CMV (230kb)
AD169T
UL US
TowneToledoPH
20 40 60 80 100 120 140 160 180 200 220 230 kb0
TRFIX
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
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
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
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
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