Epstein-Barr virus nuclear antigen 1 activates transcription from episomal but not integrated DNA and does not alter lymphocyte growth

Abstract

By binding to a cis-acting element (oriP) in the Epstein-Barr virus (EBV) genome, EBV nuclear antigen 1 (EBNA1) enables persistence and enhances transcription from EBV episomes. To investigate whether EBNA1 also directly affects cell gene transcription, we conditionally expressed a Flag-tagged dominant negative EBNA1 (FDNE) in an EBV immortalized lymphoblastoid cell line, in which the EBV genome is integrated into cell DNA. FDNE induction inhibited expression from an EBNA1-dependent oriP reporter plasmid by more than 90% in these cells but did not affect expression from integrated EBV or oriP reporter DNA. FDNE induction also did not alter expression of more than 1,800 cellular mRNAs. Lymphoblastoid cell line growth under a variety of conditions was unaffected by FDNE induction. Although Gal4-VP16 and EBNA1 strongly activated and coactivated a Gal4-VP16- and oriP-dependent promoter that was on an episome, only Gal4-VP16 activated the promoter when it was integrated into chromosomal DNA. These data indicate that EBNA1 is specifically deficient in activation of an integrated oriP enhancer and does not affect cell growth or gene expression through an interaction with cognate chromosomal DNA.

Figure 1

In A, schematic diagrams of the EBNA1 ORF, the FDNE, and the EBNA1-dependent oriP and Gal4-VP16-dependent reporter plasmids, pFL and pGFL. Basic, GA, NLS, DBD/DD, and AC refer to the basic amino acid-rich chromosome association domain, the glycine alanine-rich domain, the nuclear localization sequence, the DNA-binding and dimerization domain, and the acidic domain, respectively. In pFL and pGFL, DS, and FR refer to the dyad symmetry and family of repeats components of oriP. pFL and pGFL have a simian virus (SV)40 minimal promoter for transcription of the luciferase. The SV40 promoter initiates transcription of the puromycin acetyl transferase. In B, FDNE inhibits wild-type EBNA1 activation of luciferase expression in non-EBV-infected BJAB cells. BJAB cells were transiently cotransfected with 20 μg of pCMVEBNA1, pCMVFDNE, or pCMV, 5 μg of pFL oriP reporter, and 5 μg of pGKβ-galactosidase control DNA. In C, conditional expression of FDNE in EBV infected IB4 LCL inhibits EBNA1 activation of luciferase expression from transfected pFL plasmid. FDNE was induced or repressed for 4 days in IBDN cells, and 10 million cells were transiently cotransfected at 230 V and 65 msec with 5 μg of pFL and 2.5 μg of pGK–β-galactosidase DNA. Luciferase and β-galactosidase were assayed after 48 h. Luciferase values were corrected for transfection efficiencies as measured by β-galactosidase activity.

Figure 2

The effects of FDNE induction or repression on EBV-encoded or cell gene expression. IBDN1 and -2, subclones of IB4 LCLs with conditional FDNE expression, were plated for 4 days in complete medium with or without tetracycline to repress (−) or induce (+) FDNE, respectively. Whole cell Western blots from 2 × 10⁵ cells were done for EBNA1 and FDNE by using an EBNA1 monoclonal antibody, for all EBNAs by using an EBV immune human serum, and for c-myc by using a polyclonal rabbit antiserum. EBER and GAPDH expression was evaluated by Northern blot. A whole cell Western blot of BJAB cells that express FDNE is shown in the left lane.

Figure 3

FDNE induction inhibits EBNA1-dependent maintenance of and expression from pFL episome but does not affect expression from integrated pFL. In A, luciferase activity per 40,000 cells for each day of FDNE induction is indicated relative to the activity per 40,000 cells of the same clone grown under conditions of FDNE repression in parallel over 7 days. A subclone of IBDN1 cells with episomal pFL was compared with 11 subclones of IBDN cells with integrated pFL DNA. Representative data from triplicate experiments are shown. FDNE expression was assayed by Western blot, and results shown are for cells grown under induced conditions relative to the repressed condition at day 0 before induction. In B, the clone of IBDN cells with episomal pFL was grown under conditions of puromycin selection and FDNE induction (open circle) or repression (closed circle), and the number of viable cells was counted each day.

Figure 4

Dominant negative FDNE induction or repression does not affect LCL growth or gene expression. In A, the growth of IBDN cells in medium supplemented with 2% serum was unaffected by FDNE induction (open circles) or repression (closed squares) over 11 days. FDNE levels as assayed by Western blot are shown below the growth. In B are Northern analyses with ³²P-labeled GAPDH and EBER DNA probes of total RNA from cells 10 days after FDNE induction or repression. In C, the effect of FDNE induction for 10 days on cell gene RNA expression was evaluated by comparing RNA abundance as assayed on ≈4,100 human cDNAs under conditions of FDNE induction (EI + FDNE) and FDNE repression (EI). The ordinate and abscissa are in log base 2. The box demarcates genes whose expression is less than 2 times the median background and therefore deemed to be insignificantly different from background. The results shown are from one experiment, and the few RNAs that appear to be differentially expressed were not differentially expressed in other two experiments.

Figure 5

EBNA1 activates an EBNA1-dependent oriP episome enhancer on plasmids but not integrated into chromosomal DNA, whereas Gal4-VP16 activates a Gal4 response element on plasmids or integrated into chromosomal DNA. A shows the relative luciferase activities of 40,000 cells from a polyclonal BJAB cell line with integrated pFL grown under conditions of EBNA1 induction or repression. B shows Gal4-VP16 expression vector-mediated activation of luciferase expression from cotransfected pGL plasmid or integrated linearized pGL plasmid DNA in BJAB cells relative to expression vector control DNA. For Gal4-VP16 activation of pGL plasmid DNA, BJAB cells were cotransfected with 2 μg of pGL, 10 μg of pGal4-VP16, or vector, and 2.5 μg of pGKβ-galactosidase. For Gal4-VP16 activation of integrated DNA, polyclonal BJAB cell lines with integrated pGL DNA were transfected with 10 μg of pGal4-VP16 or vector DNA and 2.5 μg of pGKβ-galactosidase DNA. Luciferase activities are normalized for β-galactosidase and for activity with expression vector control. C shows EBNA1 and Gal4-VP16 activation of luciferase expression from pGFL plasmid or integrated DNA in polyclonal BJAB cells that have integrated linearized pGFL DNA. For EBNA1 or Gal4-VP16 activation of integrated pGFL luciferase reporter DNA, cells were cotransfected with 25 μg of pSVEBNA1, 25 μg of pGal4-VP16, and/or vector DNA to constitute 50 μg of total DNA. Luciferase activities of 2 × 10⁵ live cells were assayed at 48 h after transfection. The relative activity is normalized for vector alone. For activation from pGFL episomal plasmid DNA, the BJAB cells with integrated linearized pGFL DNA were cotransfected with 2 μg of pGFL and 2 μg of pGKβ-galactosidase DNA in addition to the 25 μg of pSVEBNA1, pGal4-VP16, or vector control DNA. The luciferase activities attributable to integrated pGFL DNA assayed in parallel were subtracted from the resulting luciferase activities, which were then normalized for the similar levels of β-galactosidase activity that were obtained and for activity with vector DNA. Data shown are the average of three experiments.