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. 2005 Aug;79(16):10171-9.
doi: 10.1128/JVI.79.16.10171-10179.2005.

Epstein-Barr virus nuclear protein 3A domains essential for growth of lymphoblasts: transcriptional regulation through RBP-Jkappa/CBF1 is critical

Seiji Maruo  1 , Eric Johannsen, Diego Illanes, Andrew Cooper, Bo Zhao, Elliott Kieff
Affiliations

Epstein-Barr virus nuclear protein 3A domains essential for growth of lymphoblasts: transcriptional regulation through RBP-Jkappa/CBF1 is critical

Seiji Maruo et al. J Virol. 2005 Aug.

Abstract

Experimental reverse genetic replacement of Epstein-Barr virus nuclear antigen 3A (EBNA3A) with a conditional mutant EBNA3A revealed that EBNA3A is critical for continued lymphoblastoid cell (LCL) growth. Wild-type (wt) EBNA3A expressed in the LCLs specifically sustained growth under nonpermissive conditions, whereas EBNA3B or EBNA3C expression had no effect (S. Mauro, E. Johannsen, D. Illanes, A. Cooper, and E. Kieff, J. Virol. 77:10437-10447, 2003). This genetic system and related biochemical assays have now been used to discover that EBNA3A lacking amino acid residues 170 to 240 (delta170-240), TLGC202 to AAGA202, or delta300-386, which are deficient in repression of EBNA2 activation of an RBP-Jkappa/CBF1-dependent EBV Cp enhancer, are null mutations for LCL growth, whereas EBNA3A delta2-124, delta410-439, delta440-470, delta470-500, delta500-523, delta523-612, and delta620-820, which are wt in repression are wt for LCL growth. Thus, EBNA3A regulation of transcription through RBP-Jkappa/CBF1 is critical for LCL growth. EBNA3A mutants deleted of amino acid residues 240 to 300, 386 to 410, or 827 to 944 were intermediate, null, or intermediate, respectively, for LCL growth despite being wt for RBP-Jkappa association and repression. Amino acid residues 240 to 300, 386 to 410, and, particularly, C-terminal residues 827 to 944 are therefore likely to contribute to LCL growth through RBP-Jkappa-independent mechanisms.

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Figures

FIG. 1.
FIG. 1.
Diagram of EBNA3A domains and mutants with summarized reverse genetic and biochemical data. The diagram indicates EBNA3A aa 146 to 155, which are the essential and sufficient nuclear localization signal (25); aa 90 to 310, which have the highest (22 to 27%) identity to other EBNA3 proteins and are designated “Homology,” T199, L200, and C202 and which are critical for RBP-Jκ association (7); aa 310 to 365, which are charged; and aa 841 to 944, which are acidic. Residues 524 to 666 and 627 to 805 have repressive and activation effects, respectively, on Gal4-responsive promoters when fused to the Gal4 DNA-binding domain (3, 4, 7, 14, 36, 37). The results of wt or mutant EBNA3A association with RBP-Jκ, repression of EBNA2 transcriptional activation of the Cp promoter and effects on LCL growth under conditions that are nonpermissive for endogenous EBNA3AHT expression are shown in columns on the right side of the figure. +, wt phenotypes; −, null; and I, intermediate. The number of transcomplementation experiments (N) performed and the number of different EBV/EBNA3AHT infected LCLs (n) used in experiments are also indicated. The parts of EBNA3A that were deleted without affecting cell growth or with an intermediate effect on cell growth are indicated at the bottom.
FIG. 2.
FIG. 2.
Transcomplementation assays of wt or mutant EBNA3As or wt EBNA3C in sustaining growth of EBV/EBNA3AHT-infected LCLs under nonpermissive conditions. (A) EBNA3AHT-infected LCL cells were transfected with 30 μg of the oriP plasmid expressing fE3A, fE3C, AAGA, Δ2-124, Δ170-240, Δ240-300, Δ300-386, Δ386-523, Δ523-612, Δ620-820, or Δ827-944, or a control oriP plasmid (Cont) and were cultured in conditioned medium with 4HT for 3 days. The cells were washed and resuspended at 1 × 106 cells/10 ml of complete medium with (+) or without (−) 4HT in 25-cm2 culture flasks (day 0). Every 5 to 7 days, cells were counted and cultures were fed with similar amounts of fresh medium. Total numbers of viable cells derived from the initial cultures were calculated and plotted at each time point. The number 10E5 along the y axis indicates that the cell number plotted should be multiplied by 100,000. (B) Protein lysates made from these cells on day 0 (3 days after transfection) were subjected to Western blotting with EBV-immune human serum to detect EBNA3A, EBNA3C, and EBNA3A mutants. (C) After 50 days of continuousculture with (+) or without (−) 4HT, protein lysates were again prepared from the transfected cells and subjected to Western blotting with EBV-immune human serum. In panels B and C, endogenous expression of EBNA3AHT (E3AHT) and EBNA3B (E3B) and expression of fE3A, fE3C, and fE3A mutants (fE3A, C, mutants) from transfected plasmids is indicated. Endogenous EBNA3C expression from the recombinant genome is not evident in blots of extracts from these LCLs because the human serum does not detect type II EBNA3C. HS, human serum.
FIG. 3.
FIG. 3.
EBNA3A aa 1 to 523 are not sufficient for maintaining LCL growth when EBNA3AHT-infected LCLs are grown under nonpermissive conditions. (A) EBNA3AHT clone 41-13 LCLs were transfected with 30 μg of the oriP plasmid expressing fE3A or FLAG-tagged EBNA3A deletion mutant (1-277, 1-386, 1-523, or Δ827-944), or a control oriP plasmid (Cont) and cultured in conditioned medium with 4HT for 3 days. The cells were washed and resuspended at 5 × 105 cells/10 ml of complete medium with (+4) or without (−) 4HT in 25-cm2 culture flasks (day 0). Every 4 to 7 days, cells were counted and cultures were fed with similar amounts of fresh medium. Total numbers of viable cells derived from the initial cultures were calculated and plotted at each time. The number 10E5 along the y axis indicates that the cell number plotted should be multiplied by 100,000. (B) Protein lysates from cells on day 0 (3 days after transfection) were subjected to Western blotting with EBV-immune human serum to detect EBNA3A and EBNA3A deletion mutants (indicated with diamonds).
FIG. 4.
FIG. 4.
EBNA3A aa 170 to 240 are required for RBP-Jκ association. (A and B) IB4 LCLs were transfected with 30 μg of the oriP plasmid expressing fE3A, fE3C, or indicated FLAG-tagged EBNA3A mutants or with a control oriP plasmid (Cont). At 48 h after transfection, protein complexes were immunoprecipitated with antibodies to FLAG and were subjected to Western blotting with FLAG- or RBP-Jκ-specific antibodies. Diamonds indicate fE3A or fE3A deletion mutants.
FIG. 5.
FIG. 5.
EBNA3A Δ170-240, AAGA202, and Δ300-386 are deficient in inhibition of EBNA2 activation of a multimerized Cp promoter. BJAB cells were transfected with the pLuc-Cp reporter construct containing eight copies of the RBP-Jκ binding site along with EBNA2 (E2+) alone or with the indicated fE3A construct. Relative luciferase activity was normalized to the β-galactosidase activity from cotransfected pGK-βgal. The results are averages from two independent experiments and are representative of three independent repetitions. Bars indicate standard errors.
FIG. 6.
FIG. 6.
EBNA3A aa 386 to 410 are essential for EBNA3A effects on LCL growth but not for RBP-Jκ association or repression of EBNA2 activation of a multimerized Cp promoter RBP-Jκ site with a luciferase reporter. (A) EBNA3AHT clone 163 LCLs were transfected with 30 μg of the oriP plasmid expressing fE3A or indicated FLAG-tagged EBNA3A mutants or with a control oriP plasmid (Cont) and cultured in conditioned medium with 4HT for 4 days. The cells were washed and suspended at 1 × 106 cells/10 ml of complete medium with (+) or without (−) 4HT in 25-cm2 culture flasks (day 0). Every 7 to 8 days, cells were counted and cultures were fed with similar amounts of fresh medium. Total numbers of viable cells derived from the initial cultures were calculated and plotted at each time point. The number 10E5 along the y axis indicates that the cell number plotted should bemultiplied by 100,000. (B) Protein lysates made from these cells on day 0 (3 days after transfection) were subjected to Western blotting with EBV-immune human serum (HS) to detect EBNA3A and EBNA3A deletion mutants. Endogenous expression of EBNA3AHT (E3AHT) and EBNA3B (E3B) and expression of fE3A and fE3A mutants (fE3A, mutants) from transfected plasmids are indicated. (C) IB4 cells were transfected with 30 μg of the oriP plasmid expressing fE3A or indicated FLAG-tagged EBNA3A deletion mutants or with a control oriP plasmid (Cont). At 48 h after transfection, protein complexes were immunoprecipitated with antibodies to FLAG and were subjected to Western blotting with FLAG- or RBP-Jκ-specific antibodies. (D) BJAB cells were transfected with the pLuc-Cp reporter construct along with EBNA2 (E2+) alone or with the indicated fE3A construct, and activation of transcription by EBNA2 was evaluated by relative luciferase activity. The results are averages of duplicate samples and are representative of two experiments with similar results. Bars indicate standard errors.
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References

    1. Abbot, S. D., M. Rowe, K. Cadwallader, A. Ricksten, J. Gordon, F. Wang, L. Rymo, and A. B. Rickinson. 1990. Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein. J. Virol. 64:2126-2134. - PMC - PubMed
    1. Alfieri, C., M. Birkenbach, and E. Kieff. 1991. Early events in Epstein-Barr virus infection of human B lymphocytes. Virology 181:595-608. (Erratum, 185: 946.) - PubMed
    1. Bourillot, P. Y., L. Waltzer, A. Sergeant, and E. Manet. 1998. Transcriptional repression by the Epstein-Barr virus EBNA3A protein tethered to DNA does not require RBP-Jkappa. J. Gen. Virol. 79:363-370. - PubMed
    1. Cludts, I., and P. J. Farrell. 1998. Multiple functions within the Epstein-Barr virus EBNA-3A protein. J. Virol. 72:1862-1869. - PMC - PubMed
    1. Cooper, A., E. Johannsen, S. Maruo, E. Cahir-McFarland, D. Illanes, D. Davidson, and E. Kieff. 2003. EBNA3A association with RBP-Jκ down-regulates c-myc and Epstein-Barr virus-transformed lymphoblast growth. J. Virol. 77:999-1010. - PMC - PubMed

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