ZEB1 regulates the latent-lytic switch in infection by Epstein-Barr virus

Abstract

The immediate-early (IE) BZLF1 gene of Epstein-Barr virus (EBV) regulates the switch between latent and lytic infection by EBV. We previously showed that the cellular transcription factor ZEB1 binds to a sequence element, ZV, located at nt -17 to -12 relative to the transcription initiation site of the BZLF1 promoter, Zp, repressing transcription from Zp in a transient transfection assay. Here, we report the phenotype in the context of a whole EBV genome of a variant of EBV strain B95.8 containing a 2-bp substitution mutation in the ZV element of Zp that reduced, but did not eliminate, ZEB1 binding to Zp. Strikingly, epithelial 293 cells latently infected with the EBV ZV mutant spontaneously produced IE-, early-, and late-gene products and infectious virus, while wild-type (WT)-infected 293 cells did not and have never been reported to do so. Furthermore, treatment with the chemical inducers sodium butyrate and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to an additional order-of-magnitude production of infectious virus in the ZV mutant-infected 293 cells, but still no virus in the WT-infected 293 cells. Similarly, ZV mutant-infected Burkitt's lymphoma BJAB cells accumulated at least 10-fold more EBV IE mRNAs than did WT-infected BJAB cells, with TPA or sodium butyrate treatment leading to an additional 5- to 10-fold accumulation of EBV IE mRNAs in the ZV mutant-infected cells. Thus, we conclude that ZEB1 binding to Zp plays a central role in regulating the latent-lytic switch in EBV-infected epithelial and B cells, suggesting ZEB1 as a target for lytic-induction therapies in EBV-associated malignancies.

Figure 1. BZLF1 Promoter

(A) Schematic indicating the cis-acting regulatory elements present in the nt −221 through +40 region of Zp. Rectangles along Zp indicate approximate locations of known regulatory elements; their trans-acting factors are indicated above them. ZEB1 concurrently binds via its two zinc-finger domains to the two cis-acting elements ZV and ZV'. The transcription initiation site is indicated by a rightward arrow. (B) Nucleotide sequence of the nt −30 to +20 region of Zp. The ZV and ZV' elements are boxed. The 2-bp substitution mutation in the ZV element shown below the sequence was introduced here into the EBV genome.

Figure 2. Presence of EBV DNA, mRNAs, and Proteins in the Latently Infected 293 Cell Lines Studied Here

(A) Southern blot analyses of the BamHI DNA fragments B, K, R, and Z of the p2089 DNAs isolated from the indicated WT- and ZV mutant-infected 293 cell lines. The locations of these BamHI fragments are indicated on the right. (B) Mutation in the Zp ZV element leads to increased accumulation of Zta and Rta mRNA in latently infected 293 cell lines. Northern blot analyses were performed to detect the steady-state levels of Zta and Rta mRNAs present in the WT- and ZVmt-infected 293 cell lines. The bands corresponding to Rta, Zta, and β-actin mRNAs are indicated on the right. Rta' corresponds to the 4-kb, amino-terminally unspliced form of Rta mRNA. The size markers indicated on the left consisted of an RNA ladder (Invitrogen) run in the same gel. (C) Mutation of the Zp ZV element leads to increased synthesis of the EBV Zta, Rta, and BMRF1 proteins. Immunoblot analyses were performed with lysates of the indicated cell lines and antisera to Zta, Rta, BMRF1, and β-actin.

Figure 3. Indirect Immunofluorescence Staining (IFS) of 293-WT Cell Line 1 and 293-ZVmt Cell Line 1 for Presence of Zta, Rta, BMRF1, and gp350 Protein

The primary antibodies used are indicated on the left of each row of images. Fields of cells were photographed with different filters to show the total EBV-positive ones (GFP) versus the subset of those containing the indicated EBV-encoded protein (IFS).

Figure 4. Production of Infectious Virus from the Latently Infected 293 Cell Lines Studied Here

(A) Green Raji units (GRUs) assay showing production of infectious virus from 293-ZVmt cell line 1, but not 293-WT cell line 1. The EBV virus released into the medium was concentrated by centrifugation and used to infect Raji cells. Fields of Raji cells were examined with visible light (visible) for total cell number versus ultraviolet light (UV) for EBV-infected, GFP-positive cells. (B) EBV termini assay for presence of linear and circular episomal EBV genomes. Plasmid DNA was isolated from 293-p2089-WT cell line 1 and 293-ZVmt cell line 1, cleaved with BamHI restriction endonuclease, separated by electrophoresis in a 0.8% agarose gel, transferred to a Hybond-N membrane (GE Healthcare), and probed with an EBV probe specific to the ends of the EBV genome [30]. The size markers indicated on the right consisted of a DNA ladder (New England Biolabs) run in the same gel. The positions of circular EBV genomes with fused termini and linear EBV genomes are indicated on the left.

Figure 5. Effect of ZV Mutation in B cell Burkitt's Lymphoma BJAB Cells

(A) Southern blot analysis of EBV DNA termini present in BJAB-WT and BJAB-ZVmt cell lines. (B) Northern blot analysis of Zta mRNA accumulated in BJAB cell lines latently infected with WT and ZVmt EBV. (C) Northern blot analysis of Zta and Rta mRNAs accumulated in BJAB-WT cell line 1 and BJAB-ZVmt cell line 1 following treatment with TPA or sodium butyrate as indicated for 48 h. The bands corresponding to Zta, Rta, and β-actin mRNAs are indicated on the right. (D) Immunofluorescence staining of BJAB-WT cell line 1 and BJAB-ZVmt cell line 1 for presence of Zta and Rta proteins. Cells were harvested and fixed 48 h after incubation in the absence or presence of TPA. Fields of cells were photographed with different filters to show the total EBV-positive ones (GFP) versus the subset of those containing the indicated EBV-encoded Zta and Rta proteins in the upper and lower panels, respectively.

Figure 6. Quantitative ChIP Assay Showing Reduced Binding of ZEB1 to Zp in ZVmt-Infected 293 Cells

(A) Autoradiogram of radiolabeled PCR products resolved by electrophoresis in a 2.5% agarose gel. Lanes 1–4, DNA templates were obtained from chromatin isolated from 293, 293-WT, 293-ZVmt, and 293-ZVmtRev cells immunoprecipitated with a ZEB1-specific antibody; and lanes 5–8, DNA templates were obtained from input DNA isolated from these cells prior to immunoprecipitation. The percent of the DNA sample used in each PCR amplification reaction is indicated on the right. (B) Summary of quantitative analysis of results of ChIP assays performed as shown in panel (A) on three separate occasions starting with cells harvested on different days. Data were normalized to the amount of PCR product obtained from 293-WT ChIP DNA and shown as means plus standard errors of the means. (C) Amplification of 1–2 μl of ChIP DNA by 25 cycles of PCR yields products in the linear range of the assay. The insert shows the autoradiogram of PCR products obtained starting with 1.0, 1.5, and 2.0 μl of ChIP DNA and the primers for Zp. (D) Autoradiogram of PCR products of DNA templates obtained by immunoprecipitation of chromatin from 293 and 293-WT cells with antibody to ZEB1 (lanes 1 and 2) versus pre-immune rabbit IgG (lanes 3 and 4). (E) Autoradiogram of PCR products from DNA templates obtained from chromatin immunoprecipitated with ZEB1 antibody (lanes 1 and 2) or input DNA isolated prior to immunoprecipitation (lanes 3 and 4) using a pair of primers corresponding to a region of EBV located 4.8-kbps upstream of the Zp transcription initiation site that lacks a ZEB1-binding site. The 190-bp PCR product is indicated by an arrow.