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Comparative Study
. 2006 Dec 19;103(51):19500-5.
doi: 10.1073/pnas.0604919104. Epub 2006 Dec 11.

Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells

Seiji Maruo  1 , Yi Wu, Satoko Ishikawa, Teru Kanda, Dai Iwakiri, Kenzo Takada
Affiliations
Comparative Study

Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells

Seiji Maruo et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C-HT. In the presence of 4HT, LCLs expressed the E3C-HT protein and grew like WT LCLs. When E3C-HT EBV-infected LCLs were transferred to medium without 4HT, E3C-HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C-HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16INK4A, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G2/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Establishment of LCLs infected with recombinant EBV expressing E3C–HT. (A) Construction of the E3C–HT EBV bacmid. A schematic representation of the EBV genomic EBNA3 region of AK-BAC-GFP is shown. A DNA construct containing the 4HT-responsive mutant estrogen receptor hormone-binding domain (HTER) and a zeocin marker (zeo) flanked by mutant loxP sites was inserted downstream of the EBNA3C ORF in AK-BAC-GFP by GET recombination. The zeocin marker was removed in vitro by treatment with Cre recombinase. The positions of PCR primers are indicated by arrows. (B) PCR analysis of the EBNA3B and EBNA3C genotypes of E3C–HT LCLs. The specific primer pairs distinguish between EBV type 1 and type 2 EBNA3B (E3B) and EBNA3C (E3C), and primers flanking the junction between the EBNA3C and HTER genes (E3C–HT) were used for amplification of fragments from type 1 WT LCLs, type 2 P3HR-1 cells, and four independent E3C–HT LCLs. Control amplifications without template are also shown (Primers). The E3B primers amplify 125- or 149-bp fragments from type 1 or type 2 DNA, respectively. The E3C primers amplify 153- or 246-bp fragments from type 1 or type 2 DNA, respectively. (C) E3C–HT LCLs express a large E3C–HT fusion protein and do not express WT EBNA3C. Total cell lysates of two independent WT LCLs and four independent E3C–HT LCLs were tested for the expression of EBNAs by Western analysis with EBV-immune human serum.
Fig. 2.
Fig. 2.
EBNA3C is essential for LCL growth maintenance. (A) The E3C–HT protein slowly disappears after LCLs are transferred to medium without 4HT. Total cell lysates of E3C–HT LCLs, cultured in the absence (−) or presence (+) of 4HT for the indicated number of days, were immunoblotted with EBV-immune human serum or with an actin antibody. (B) E3C–HT LCLs depend on 4HT for growth. WT LCLs and four independent E3C–HT LCLs were cultured with (+) or without (−) 4HT. Viable cell numbers were determined by trypan blue exclusion, and total viable cell numbers derived from initial cell cultures are plotted. (C) Four independent E3C–HT LCLs were cultured in the absence (−) or presence (+) of 4HT for 17 days. Total cell lysates were prepared and subjected to Western blot analysis. (D) WT EBNA3C expression sustains the growth of E3C–HT LCLs in the absence of 4HT. E3C–HT LCLs were transfected with an oriP plasmid expressing EBNA3A (E3A), EBNA3B (E3B), EBNA3C (E3C), or a control oriP plasmid (Cont.). Five days after transfection (day 0), cells were washed and transferred to medium with (+) or without (−) 4HT, and cell growth was determined as described in B. (E) E3C–HT LCLs express higher levels of transfected WT EBNA3C in the absence of 4HT than in its presence. Total cell lysates were prepared from the cultures shown in D at the indicated time points and tested for EBNA3 expression by Western blot analysis with EBV-immune human serum. Note that transfected WT EBNA3C is derived from the EBV strain B95-8 and therefore migrates between EBNA3A and EBNA3B on an SDS/PAGE gel. HS blot, EBV-immune human serum blot. (F) Total cell lysates were prepared from the cultures shown in D at day 16 and subjected to Western blot analysis.
Fig. 3.
Fig. 3.
E3C–HT inactivation does not affect the expression of other EBNAs, LMP1, c-myc, CD21, or CD23. (A) E3C–HT LCLs were cultured in the absence (−) or presence (+) of 4HT. Total cell lysates were prepared at the indicated time points and analyzed by Western blot with EBV-immune human serum or LMP1-, c-myc-, or actin-specific antibodies. (B) E3C–HT LCLs were cultured with (+) or without (−) 4HT for 14 days. The cell surface expression of CD21 and CD23 was monitored by flow cytometry (solid lines). Staining with isotype-matched control antibody is indicated by dotted lines. Each experiment was repeated with two independent E3C–HT LCLs with similar results.
Fig. 4.
Fig. 4.
EBNA3C inactivation results in a decrease in the proportion of cells in S and G2/M phases, which is accompanied by a decrease in the level of hyperphosphorylated pRb. (A) E3C–HT LCLs were cultured in medium without 4HT for the indicated number of days. Cells were fixed and stained with propidium iodide, and cell cycle analysis was performed with a FACSCalibur. (B) E3C–HT LCLs were cultured in the absence (−) or presence (+) of 4HT for the indicated number of days. Total cell lysates were prepared, and Western blot analysis was performed with Rb- or actin-specific antibodies. Each experiment was repeated with two independent E3C–HT LCLs with similar results.
Fig. 5.
Fig. 5.
EBNA3C inactivation results in the accumulation of p16INK4A. (A) EBNA3C inactivation results in an increase in the level of p16 protein in LCLs. E3C–HT LCLs were cultured with (+) or without (−) 4HT for the indicated number of days. Western blot analysis with p16-, p21-, p27-, cyclin A (cycA)-, or actin-specific antibodies was performed. (B) Transfection of WT EBNA3C into E3C–HT LCLs prevents the accumulation of p16 in the absence of 4HT. E3C–HT LCLs were transfected with an oriP plasmid expressing WT EBNA3C (E3C) or a control oriP plasmid (Cont). Cells were cultured in medium with (+) or without (−) 4HT for 21 days and subjected to Western blot analysis with p16- and actin-specific antibodies. (C) EBNA3C inactivation results in an increase in the level of p16 transcript. Total RNA was extracted from E3C–HT LCLs cultured in medium with (+) or without (−) 4HT for 6 or 11 days. Real-time RT-PCR for p16 was performed, and the results were normalized against GAPDH. Reactions were done in triplicate. (D) EBNA3C inactivation results in an increase in the level of the p16/Cdk4 complex and a decrease in the level of the cyclin D2/Cdk4 complex. E3C–HT LCLs cultured with (+) or without (−) 4HT for 20 days were lysed and immunoprecipitated with a Cdk4-specific antibody. Total cell lysates were subjected to Western blot analysis with Cdk4-, Cdk6-, cyclin D2 (cycD2)-, and actin-specific antibodies. Immunoprecipitated samples (Cdk4 IP) were resolved by SDS/PAGE and blotted with Cdk4-, p16-, and cyclin D2-specific antibodies. Each experiment was repeated with two independent E3C–HT LCLs with similar results.
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