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.2010 Jun;84(12):6139-52.
doi: 10.1128/JVI.02706-09. Epub 2010 Apr 7.

Either ZEB1 or ZEB2/SIP1 can play a central role in regulating the Epstein-Barr virus latent-lytic switch in a cell-type-specific manner

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Either ZEB1 or ZEB2/SIP1 can play a central role in regulating the Epstein-Barr virus latent-lytic switch in a cell-type-specific manner

Amy L Ellis et al. J Virol.2010 Jun.

Abstract

We previously reported that the cellular protein ZEB1 can repress expression of the Epstein-Barr virus (EBV) BZLF1 gene in transient transfection assays by directly binding its promoter, Zp. We also reported that EBV containing a 2-bp substitution mutation in the ZEB-binding ZV element of Zp spontaneously reactivated out of latency into lytic replication at a higher frequency than did wild-type EBV. Here, using small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies, we definitively show that ZEB1 is, indeed, a key player in maintaining EBV latency in some epithelial and B-lymphocytic cell lines. However, in other EBV-positive epithelial and B-cell lines, another zinc finger E-box-binding protein, ZEB2/SIP1, is the key player. Both ZEB1 and ZEB2 can bind Zp via the ZV element. In EBV-positive cells containing only ZEB1, knockdown of ZEB1 led to viral reactivation out of latency, with synthesis of EBV immediate-early and early lytic gene products. However, in EBV-positive cells containing both ZEBs, ZEB2, not ZEB1, was the primary ZEB family member bound to Zp. Knockdown of ZEB2, but not ZEB1, led to EBV lytic reactivation. Thus, we conclude that either ZEB1 or ZEB2 can play a central role in the maintenance of EBV latency, doing so in a cell-type-dependent manner.

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Figures

FIG. 1.
FIG. 1.
Schematic diagrams showing structures of ZEB1 and ZEB2 proteins (A), promoter region of theBZLF1 gene indicating itscis-acting elements and theirtrans-acting factors (B), and the sequences of the wild-type (WT) and ZV mutant (mt) DNA probes used in the EMSAs (C). The ZV and ZV′ elements are indicated by boxes in panel C, with the 2-bp substitution mutation in the ZV element indicated by italicized, underlined letters. SID, Smad-interacting domain; CID, CtBP-interacting domain.
FIG. 2.
FIG. 2.
Both ZEB1 and ZEB2 repress transcription of Zp via binding the ZV element. (A) EMSAs showing ZEB1 and ZEB2 binding to Zp. ZEB1 and ZEB2, synthesized in a cell-free system (TNT), were incubated with a radiolabeled double-stranded WT DNA corresponding to the nt −30 to +20 region of Zp (Fig. 1C) as a probe in the presence or absence of antibody specific to ZEB1 or ZEB2. Lanes 1 to 3, EMSAs performed in parallel with unprogrammed reticulocyte (Retic.) lysate as a negative control. (B and C) Competition EMSAs showing ZEB1 and ZEB2 bind via the ZV element to Zp. Extracts prepared from 293D cells transiently transfected with plasmids expressing ZEB1 (B) or ZEB2 (C) were incubated with the same radiolabeled DNA probe used in panel A together with a 0, 20, 40, or 80 M excess of nonradiolabeled, double-stranded DNA corresponding to the WT or ZVmt sequence shown in Fig. 1C. (D) MCF-7 cells were cotransfected as indicated with 0.5 μg of the reporter plasmid pZpWT-luc or pZpZVmt-luc (i), 0.5 μg of pCMXRluc as an internal control (ii), and 0.05 μg of an expression plasmid encoding ZEB1, ZEB2, or their empty parental vector, pcDNAhismaxC (iii). Forty-eight hours later, the cells were harvested, and relative (Rel.) luciferase activity was measured. Data were internally normalized to the value for renilla luciferase present in the same sample and externally normalized to the value obtained for the cells cotransfected with pZpWT-luc, pCMXRluc, and pcDNA4hismaxC. Shown here is a representative experiment indicating the means ± standard errors of the means from assays performed in triplicate. (E) Immunoblot analysis of MCF-7 cells transfected as described for panel D with 0.05 μg of a plasmid expressing ZEB1, ZEB2, or the empty vector pcDNA4hismaxC as a control. The cells were harvested 48 h after transfection, and 30 μg of whole-cell protein was loaded per lane and analyzed as described in Materials and Methods for ZEB1 and ZEB2 protein, with β-actin serving as a loading control. Lane 1 was loaded with 30 μg of whole-cell protein obtained from BJABB95.8 cells as a control for endogenous levels of ZEB1 and ZEB2 protein present in latently infected cells.
FIG. 3.
FIG. 3.
ZEB1 and ZEB2 levels vary in different EBV-positive cell lines. (A and B) Immunoblots showing levels of endogenous cellular ZEB1 and ZEB2 and lytic EBV-encoded BZLF1 and BMRF1 proteins present in a variety of EBV-positive B-lymphocytic (A) and epithelial (B) cell lines. Whole-cell extracts were prepared from the indicated cell lines. Proteins (30 μg per sample) were separated by SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antisera as described in Materials and Methods. β-Actin served as a loading control. (C) RT-qPCR analysis of relative levels of ZEB1 and ZEB2 RNA present in a variety of EBV-positive cell lines. Samples were processed as described in Materials and Methods. Data were normalized to values for ribosomal Po RNA present in the same samples.
FIG. 4.
FIG. 4.
ZEB1 and ZEB2 expression correlate with EBV latency. (A and B) Scatter plots showing correlation between level of BZLF1 RNA and level of ZEB1 (A) or ZEB2 (B) RNA in EBV-positive cell lines. Relative BZLF1, ZEB1, and ZEB2 RNA levels were determined by RT-qPCR as described in Materials and Methods. Data were normalized to values for ribosomal Po RNA present in the same samples.
FIG. 5.
FIG. 5.
Knockdown of ZEB1 leads to EBV lytic reactivation in ZEB2-negative cell lines. (A) Immunoblot showing effects of siRNAs to ZEB1 and ZEB2 on maintenance of EBV latency in epithelial NPC CNE1Akata cells. CNE1 cells latently infected with the Akata strain of EBV were transfected with 30 nM the indicated siRNAs. Experiments were also performed using an alternative ZEB2 siRNA (indicated as ZEB2a) as indicated in Materials and Methods. Seventy-two hours later, whole-cell extracts were prepared, and protein levels were determined by immunoblot analysis. β-Actin served as a loading control. (B) Immunoblot showing effects of shRNAs to ZEB1 and ZEB2 on maintenance of EBV latency in EBV-positive BL MutuI cells. MutuI cells were infected with lentiviruses expressing the indicated shRNAs as described in Materials and Methods. Experiments were performed using an alternative ZEB2 shRNA (indicated as ZEB2a) as well. The cells were incubated, harvested, and analyzed as for panel A.
FIG. 6.
FIG. 6.
Immunoblots showing that overexpression of either ZEB1 (A and B) or ZEB2 (C and D) can inhibit induction of EBV lytic reactivation. (A and C) ZEB2-negative CNE1Akata cells were transfected with 1.0 μg of ZEB1 (A) or ZEB2 (C) expression plasmid or their empty parental plasmid, pcDNAhismaxC (−), as a control. Twenty-four hours later, the cells were transfected with 30 nM either ZEB1 siRNAs (+) or scrambled siRNA (−) as a control. Whole-cell extracts were prepared 72 h later and assayed for the indicated proteins by immunoblot analysis. (B and D) CNE1Akata cells were cotransfected with 0, 0.1, 0.25, 0.5, or 1.0 μg of ZEB1 (B) or ZEB2 (D) expression plasmid along with pcDNAhismaxC DNA to apply 1.0 μg total DNA per well in a 6-well plate. Twenty-four hours later, TPA (20 ng/ml) and sodium butyrate (3 mM) were added to the medium and incubation was continued for an additional 24 h prior to harvesting for whole-cell protein and assaying for the indicated proteins by immunoblot analysis. β-Actin served as a loading control.
FIG. 7.
FIG. 7.
ZEB2 is the major ZEB protein maintaining EBV latency in ZEB1+, ZEB2+ cells. (A) Immunoblots showing extent of lytic replication after treatment of 293B95.8 cells with various combinations of inducers. 293B95.8 cells were transfected with 30 nM the indicated siRNAs. Twenty-four hours later, we added to the medium dimethyl sulfoxide (DMSO) (lanes 2 to 5), the solvent for the chemicals, to 0.1% or TPA and sodium butyrate (lanes 6 to 9) to final concentrations of 20 ng/ml and 3 mM, respectively, and continued incubating the cells for an additional 48 h. Whole-cell extracts were prepared and the indicated proteins were assayed by immunoblot analysis. β-Actin served as the loading control. The positive-control (Pos. cont.) lane contained protein from cells reactivated by transfection with a BZLF1 expression plasmid. (B) Immunoblots showing extent of lytic replication after treatment of BJABB95.8 cells with various combinations of inducers. B-lymphocytic BJABB95.8 cells were infected with lentiviruses expressing the indicated shRNAs. Twenty-four hours later, DMSO (lanes 2 to 5) or TPA plus butyrate (lanes 6 to 9) was added to the medium, and the cells were subsequently treated as described for panel A. (C and D) Analysis of alternative ZEB2 siRNAs and shRNAs in EBV-positive cell lines. (C) 293B95.8 cells were transfected with 30 nM siRNAs to ZEB1, to an alternative ZEB2 (indicated as ZEB2a), or to both ZEBs or with negative-control scrambled siRNA. Experiments were performed identically to those described for panel A. (D) BJABB95.8 cells were infected with lentiviruses expressing shRNAs to ZEB1, an alternative ZEB2 (indicated as ZEB2a), both ZEBs, or negative-control pLKO.1, and experiments were performed identically to those described for panel B.
FIG. 8.
FIG. 8.
Confirmation that ZEB2, not ZEB1, is the major ZEB protein inhibiting EBV lytic reactivation in ZEB1+, ZEB2+ cells. (A and B) Immunoblots showing extent of inhibition of lytic reactivation by overexpression of ZEB1 (A) and ZEB2 (B) in 293B95.8 cells subsequently induced by a combination of the indicated siRNAs and TPA plus butyrate. 293B95.8 cells were transfected with 1.0 μg of ZEB1 (A), ZEB2 (B), or their empty parental expression plasmid (−). Twenty-four hours later, the cells were transfected with 30 nM the indicated siRNAs, incubated in the presence of TPA plus butyrate, and processed as described in the legend to Fig. 7. Exp., exposure. (C and D) Immunoblots showing extent of inhibition of lytic reactivation by overexpressed ZEB1 (C) versus ZEB2 (D) in 293B95.8 cells induced with BZLF1 protein. 293B95.8 cells were transfected with 0, 0.1, 0.25, 0.5, or 1.0 μg of an expression plasmid encoding ZEB1 (C) or ZEB2 (D), along with 1.0, 0.9 0.75, 0.5, or 0 μg of pcDNAhismaxC parental vector to allow for equal amounts of transfected DNA. Twenty-four hours later, cells were transfected with 30 nM siRNAs to ZEB2, incubated in the presence of TPA plus butyrate, and processed as described in the legend to Fig. 7. Fold changes in lytic protein expression were determined by densitometry, with internal normalization to β-actin and external normalization to samples containing no ZEBs (lane 1).
FIG. 9.
FIG. 9.
ZEB1 and ZEB2 compete for binding to Zpin vitro. EMSAs in which ZEB1 and ZEB2, synthesized in a cell-free system (TNT), were incubated with a radiolabeled double-stranded WT DNA corresponding to the nt −30 to +20 region of Zp. Each lane contained the same amount of ZEB1 protein. Lanes 2 to 7 also contained ZEB2 protein such that the ratios of ZEB1:ZEB2 for lanes 2 to 6 were 4:1, 2:1, 1:1, 1:2, and 1:4, respectively, with unprogrammed reticulocyte lysate added as well to ensure that each reaction mixture contained the same total amount of protein lysate. Lane 7, the reaction mixture was identical to the one in lane 6 except for the inclusion as well of antibody specific to ZEB2. ZEB, truncated version of ZEB2.
FIG. 10.
FIG. 10.
ZEB1 and ZEB2 compete for binding to Zpin vivo. (A to D) ChIP assays indicating relative (Rel.) binding of ZEB1 and ZEB2 to Zp in ZEB2-positive (A and B) versus ZEB2-negative (C and D) cell lines. Shown here are UV light photographs of ethidium bromide-stained agarose gels containing the PCR products obtained from ChIP assays performed as described in Materials and Methods with the indicated antisera on chromatin obtained from EBV-positive 293B95.8 cells (A), BJABB95.8 cells (B), CNE1Akata cells (C), and MutuI cells (D). Binding of ZEB1 and ZEB2 was determined for the ZV region of Zp within the EBV genomes, a ZEB-binding region of the interleukin-2 (IL-2) promoter as a positive control, and a sequence within these EBV genomes located 5 kbp upstream of Zp as a negative control (Neg. Cont.). Antibodies to IgG were used as a negative control. (E to H) ChIP-assayed samples from panels A to D above were also analyzed by qPCR for binding of ZEB2 to Zp in 293B95.8 cells (E), BJABB95.8 cells (F), CNE1Akata cells (G), and MutuI cells (H), respectively. Data were normalized first to the percentage of input DNA and then to the anti-IgG control.
FIG. 11.
FIG. 11.
ZEB2 binding to Zpin vivo is dependent upon presence of the ZV element. (A) ChIP assays showing relative binding of ZEB2 to Zp in 293B95.8 (WT) versus 293ZVmt (MT) cells. Shown here are UV light photographs of ethidium bromide-stained agarose gels containing the PCR products obtained from chromatin immunoprecipitation assays performed as described in Materials and Methods with the indicated antisera and chromatin from EBV-positive 293B95.8 cells (lanes 1, 3, and 5) or 293ZVmt cells (lanes 2, 4, and 6). Binding of ZEB2 was determined for the ZV region of Zp, the IL-2 promoter as a positive control, and a region 5 kbp upstream of Zp as a negative control (Neg. Cont.). (B) ChIP-assayed samples from panel A above were also analyzed by qPCR for binding of ZEB2 to the Zp region of the EBV genomes present in 293B95.8 cells versus 293ZVmt cells. Data were normalized to values for both the input DNA and the anti-IgG control with WT EBV.
FIG. 12.
FIG. 12.
Working model for how ZEB1 and ZEB2 contribute to regulatingBZLF1 gene expression. See Discussion for details. A, activator; CoRep, corepressor; CoAct, coactivator.
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