ZEB1 Regulates the Latent- -Lytic Lytic Switch Switch ZEB1 - - PowerPoint PPT Presentation

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ZEB1 Regulates the Latent ZEB1 Regulates the Latent-

• Lytic

Lytic Switch Switch in Infection by Epstein in Infection by Epstein-

• Barr Virus

Barr Virus

Xianming Yu, Zhenxun Wang, Janet E. Mertz PLoS Pathogen, 3(12): 867-77

Speaker: Speaker: Chien Chien-

• Sin Chen

Sin Chen Advisor: Li Advisor: Li-

• Kwan Chang, Ph.D.

Kwan Chang, Ph.D.

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Epstein Epstein-

• Barr Virus (EBV)

Barr Virus (EBV)

A kind of dsDNA γ-herpesvirus Capsid diameter: approx. 100 nm

Host:

Epithelial cells and B lymphocytes (293 cell & BJAB cell)

Life cycle:

Latent/lytic 2 stage

Pathogenicity:

90% people was infected Hodgkin’s disease, Burkitt’s lymphoma, and nasopharyngeal carcinoma

http://herpesvirus.bioworldantibodies.com/gallery/gallery.htm http://www.virology.net/Big_Virology/BVDNAherpes.html

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Life cycle of EBV Life cycle of EBV

Latent phase Lytic phase

Chemical inducers (TPA, Na butyrate), Stress

Reactivation

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Lytic Lytic cascade cascade

Early (E) genes BMRF1, BSLF1, BGLF4, … replication machineries, kinases, … Late (L) genes BLLF1, BDLF1, … glycoproteins, capsid proteins, … Immediate-early (IE) genes BZLF1, BRLF1 transcription factors

(Prof. Chang’s virology ppt)

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BZLF1 ( BZLF1 (Zta Zta) )

(Mol. Cell 2006 C Petosa et al) (Mol. Cell 2006 C Petosa et al)

• riLyt

EBV lyitc replication orgin Lytic gene activation Cellular gene regulation AP-1 site

Zta ZRE

bZIP AP-1 like

p21 Cell cycle arrest

Cell cycle machinery

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The BZLF1 promoter ( The BZLF1 promoter (Zp Zp) )

• The promoter of

The promoter of Zta Zta ( (Zp Zp) is regulated by viral ) is regulated by viral and cellular proteins and cellular proteins

• Zta

Zta promoter: promoter:

(J. Virol. 2003 RJ Kraus et al)

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Zinc finger E Zinc finger E-

• box binding

box binding homeobox homeobox 1 1 (ZEB1) (ZEB1)

• Function:

Function:

Recruit repressing complex Recruit repressing complex Play a role in differentiation Play a role in differentiation

• An example:

An example:

Growth hormone gene Growth hormone gene

ZEB1RE ZEB1RE ZEB1 Transcription complex (LSD1 complex)

Gh Gh

ZEB1 Demethylase activity

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ZEB1 suppress ZEB1 suppress Zp Zp activity activity

Mutation in ZEB1 binding site

(J. Virol. 2003 RJ Kraus et al)

From reporter assay to recombinant virus modeling

Zta ZV ZEB1

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Recombinant EBV Recombinant EBV

• In 1998, recombinant EBV

In 1998, recombinant EBV made debut made debut

• It contains

It contains

GFP tag GFP tag Hygromycin Hygromycinr

r gene

gene EBV B95.8 genome EBV B95.8 genome F factor F factor

• Why is it so good?

Why is it so good?

Can be manipulated in Can be manipulated in E. coli

• E. coli

Infect natural hosts Infect natural hosts Propagate like wild Propagate like wild-

• type B95.8

type B95.8 EBV EBV

(Proc. Natl. Acad. Sci. USA 1998 HJ Delecluse et al)

GFP Total infected cell Viral Capsid Antigen (VCA) A lytic protein Cells contain reactivated virus

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The specific aim The specific aim

F factor Hygr GFP Zta ZV Zta ZV Zta ZVmt Zta ZVmt Zta ZEB1 ZEB1 ZEB1 ZEB1 Zp

?

Recombinant viral genome

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Construction of recombinant EBV Construction of recombinant EBV

WT ZVmt ZVmtRev

F factor Hygr Zp GFP

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Framework Framework

ZEB1 Zta ZVmt

Spontaneous reactivation

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Reactivation Susceptibility to chemical inducers

ZEB1 Zta ZVmt

? ?

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Framework Framework

ZEB1 Zta ZVmt

Spontaneous reactivation

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Reactivation Susceptibility to chemical inducers

ZEB1 Zta ZVmt

? ?

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ZVmt ZVmt EBV spontaneously reactivate EBV spontaneously reactivate

mRNA level Protein level

In 293 cell

Rta & Zta: immediate-early (IE) genes express first once EBV enter lytic stage BMRF1: one of early genes, activate just after IE genes express

Lytic cascade: IE E L Reactivation

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ZVmt ZVmt EBV spontaneously reactivate EBV spontaneously reactivate

IE gene E gene IE gene L gene

In 293 cell

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ZVmt ZVmt EBV spontaneously reactivate EBV spontaneously reactivate

In BJAB cell

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but but… …, does that mutant , does that mutant produce produce virion virion finally? finally?

Zta ZVmt Zta ZVmt ZEB1 ZEB1

Lytic phase

seems TRUE so far

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Framework Framework

ZEB1 Zta ZVmt

Spontaneous reactivation

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?

Reactivation Susceptibility to chemical inducers

?

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ZEB1 Zta ZVmt

?

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ZVmt ZVmt EBV DOES produce EBV DOES produce virion virion

Stable episome Rolling circle DNA replication Concatemeric DNA: sets of viral genome

293 Virion from medium Raji

UV detection

Single set

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ZVmt ZVmt EBV is more susceptible to EBV is more susceptible to chemical inducers chemical inducers

UV detection

Raji Virion from medium 293 treatment

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ZVmt ZVmt EBV does NOT produce EBV does NOT produce virion virion

In BJAB cell

No linear small-sized DNA segment is detected

Viral genome replication is NOT observed

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ZVmt ZVmt EBV is more susceptible to EBV is more susceptible to chemical inducers chemical inducers

In BJAB cell

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And And… …, is mutation of ZV with , is mutation of ZV with weaker binding ability to ZEB1? weaker binding ability to ZEB1?

Zta ZVmt Zta ZVmt ZEB1 ZEB1

Lytic phase

s e e m s T R U E s

• f

a r 2 9 3 c e l l V B J A B c e l l X

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Framework Framework

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ZEB1 Zta ZVmt

?

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Reactivation Susceptibility to chemical inducers

?

ZEB1 Zta ZVmt

Spontaneous reactivation

1

?

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ZVmt ZVmt DOES reduce ZEB1 binding DOES reduce ZEB1 binding ability to ability to Zp Zp

ChIP: Chromatin Immunoprecipitation

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SUMMARY SUMMARY

Zta ZVmt Zta ZVmt ZEB1 ZEB1

Lytic phase

Zta ZV Zta ZV ZEB1 ZEB1 293 V BJAB V 293 V BJAB V 293 V BJAB X

Cell line

ZEB1 is very important to EBV life cycle switch, which could be a target for drug design to EBV related diseases

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Thanks for your attention! Thanks for your attention!