ZEB1 Regulates the Latent- -Lytic Lytic Switch Switch ZEB1 Regulates the Latent in Infection by Epstein- -Barr Virus Barr Virus in Infection by Epstein Xianming Yu, Zhenxun Wang, Janet E. Mertz PLoS Pathogen, 3(12): 867-77 Speaker: Chien Chien- -Sin Chen Sin Chen Speaker: Advisor: Li- -Kwan Chang, Ph.D. Kwan Chang, Ph.D. Advisor: Li 1 1
Epstein- -Barr Virus (EBV) Barr Virus (EBV) Epstein A kind of dsDNA γ -herpesvirus Capsid diameter: approx. 100 nm Host: Epithelial cells and B lymphocytes (293 cell & BJAB cell) http://www.virology.net/Big_Virology/BVDNAherpes.html Life cycle: Latent/lytic 2 stage Pathogenicity: 90% people was infected Hodgkin’s disease, Burkitt’s lymphoma, and nasopharyngeal carcinoma 2 2 http://herpesvirus.bioworldantibodies.com/gallery/gallery.htm
Latent phase 3 3 Life cycle of EBV Life cycle of EBV Lytic phase Reactivation (TPA, Na butyrate), Chemical inducers Stress
Lytic cascade cascade Lytic Immediate-early (IE) genes BZLF1, BRLF1 transcription factors Early (E) genes BMRF1, BSLF1, BGLF4, … replication machineries, kinases, … Late (L) genes BLLF1, BDLF1, … glycoproteins, capsid proteins, … (Prof. Chang’s virology ppt) 4 4
BZLF1 (Zta Zta) ) BZLF1 ( AP-1 like p21 Cell cycle machinery bZIP Cell cycle arrest Zta ZRE AP-1 site Lytic gene activation ( Mol. Cell 2006 C Petosa et al) Cellular gene regulation oriLyt EBV lyitc replication orgin ( Mol. Cell 2006 C Petosa et al) 5 5
The BZLF1 promoter (Zp Zp) ) The BZLF1 promoter ( � 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) 6 6
Zinc finger E- -box binding box binding homeobox homeobox 1 1 Zinc finger E (ZEB1) (ZEB1) ZEB1 ZEB1RE � Function: Function: � Gh Recruit repressing complex Recruit repressing complex Play a role in differentiation Play a role in differentiation Transcription An example: � An example: � complex Growth hormone gene Growth hormone gene (LSD1 complex) Demethylase activity ZEB1 ZEB1RE Gh 7 7
recombinant virus modeling 8 8 Zta From reporter assay Zp activity activity to (J. Virol. 2003 RJ Kraus et al) ZV ZEB1 ZEB1 suppress Zp ZEB1 suppress Mutation in ZEB1 binding site
Recombinant EBV Recombinant EBV � In 1998, recombinant EBV In 1998, recombinant EBV � GFP Total infected cell made debut made debut � It contains It contains � GFP tag GFP tag r gene Hygromycin r gene Hygromycin EBV B95.8 genome EBV B95.8 genome F factor F factor Viral Capsid Antigen (VCA) � Why is it so good? Why is it so good? � A lytic protein Can be manipulated in E. coli Can be manipulated in E. coli Infect natural hosts Infect natural hosts Cells contain Propagate like wild- -type B95.8 type B95.8 Propagate like wild reactivated virus EBV EBV (Proc. Natl. Acad. Sci. USA 1998 HJ Delecluse et al) 9 9
� Zta 10 10 Recombinant viral genome Zta Zta The specific aim The specific aim ZVmt ZV ZEB1 ZEB1 ? GFP Zp Hyg r F factor Zta Zta ZEB1 ZEB1 ZVmt ZV
Construction of recombinant EBV Construction of recombinant EBV F factor Hyg r WT GFP Zp ZVmt ZVmtRev 11 11
Framework Framework ZEB1 ? Spontaneous Zta 1 ZVmt reactivation Reactivation ? 2 Susceptibility to chemical inducers ZEB1 ? 3 Zta ZVmt 12 12
Framework Framework ZEB1 ? Spontaneous Zta 1 ZVmt reactivation Reactivation ? 2 Susceptibility to chemical inducers ZEB1 ? 3 Zta ZVmt 13 13
ZVmt EBV spontaneously reactivate EBV spontaneously reactivate ZVmt In 293 cell mRNA level Rta & Zta: immediate-early (IE) genes express first once EBV enter lytic stage Lytic cascade: Reactivation IE E L Protein level BMRF1: one of early genes, activate just after IE genes express 14 14
ZVmt EBV spontaneously reactivate EBV spontaneously reactivate ZVmt In 293 cell IE gene IE gene E gene L gene 15 15
ZVmt EBV spontaneously reactivate EBV spontaneously reactivate ZVmt In BJAB cell 16 16
seems TRUE ZEB1 ZEB1 so far Zta Zta ZVmt ZVmt Lytic phase but… …, does that mutant , does that mutant but produce virion virion finally? finally? produce 17 17
Framework Framework ZEB1 ? Spontaneous Zta 1 ZVmt reactivation Reactivation ? 2 Susceptibility to chemical inducers ZEB1 ? 3 Zta ZVmt 18 18
ZVmt EBV DOES produce EBV DOES produce virion virion ZVmt 293 Virion from medium Raji UV detection Rolling circle DNA replication Single set Stable episome Concatemeric DNA: sets of viral genome 19 19
ZVmt EBV is more susceptible to EBV is more susceptible to ZVmt chemical inducers chemical inducers treatment 293 Virion from medium Raji UV detection 20 20
ZVmt EBV does NOT produce EBV does NOT produce virion virion ZVmt In BJAB cell No linear small-sized DNA segment is detected Viral genome replication is NOT observed 21 21
ZVmt EBV is more susceptible to EBV is more susceptible to 22 22 chemical inducers chemical inducers In BJAB cell ZVmt
E U R T s ZEB1 m e e s ZEB1 r Zta a Zta f o ZVmt ZVmt s V l l e X c Lytic phase l 3 l e 9 c 2 B A J B And… …, is mutation of ZV with , is mutation of ZV with And weaker binding ability to ZEB1? weaker binding ability to ZEB1? 23 23
Framework Framework ZEB1 ? Spontaneous Zta 1 ZVmt reactivation Reactivation ? 2 Susceptibility to chemical inducers ZEB1 ? 3 Zta ZVmt 24 24
ZVmt DOES reduce ZEB1 binding DOES reduce ZEB1 binding ZVmt ability to Zp Zp ability to ChIP: Chromatin Immunoprecipitation 25 25
SUMMARY SUMMARY ZEB1 Cell line ZEB1 Zta Zta 293 V ZV ZV BJAB V ZEB1 ZEB1 Zta Zta ZVmt ZVmt 293 V BJAB V Lytic phase ZEB1 is very important to EBV life cycle switch, which could be a target for drug design to EBV related diseases 293 V BJAB X 26 26
27 27 Thanks for your attention! Thanks for your attention!
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