Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 May;78(10):4983-92.
doi: 10.1128/jvi.78.10.4983-4992.2004.

The BRRF1 early gene of Epstein-Barr virus encodes a transcription factor that enhances induction of lytic infection by BRLF1

Gregory K Hong  1 , Henri-Jacques Delecluse, Henri Gruffat, Thomas E Morrison, Wen-Hai Feng, Alain Sergeant, Shannon C Kenney
Affiliations

The BRRF1 early gene of Epstein-Barr virus encodes a transcription factor that enhances induction of lytic infection by BRLF1

Gregory K Hong et al. J Virol. 2004 May.

Abstract

The switch from the latent to the lytic form of Epstein-Barr virus (EBV) infection is mediated by expression of the viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). An EBV early protein, BRRF1 (Na), is encoded by the opposite strand of the BRLF1 intron, but the function of this nuclear protein in the viral life cycle is unknown. Here we demonstrate that Na enhances the R-mediated induction of lytic EBV infection in 293 cells latently infected with a recombinant EBV (R-KO) defective for the expression of both R and Na. Na also enhances R-induced lytic infections in a gastric carcinoma line (AGS) carrying the R-KO virus, although it has no effect in a Burkitt lymphoma line (BL-30) stably infected with the same mutant virus. We show that Na is a transcription factor that increases the ability of R to activate Z expression from the R-KO viral genome in 293 cells and that Na by itself activates the Z promoter (Zp) in EBV-negative cells. Na activation of Zp requires a CRE motif (ZII), and a consensus CRE motif is sufficient to transfer Na responsiveness to the heterologous E1b promoter. Furthermore, we show that Na enhances the transactivator function of a Gal4-c-Jun fusion protein but does not increase the transactivator function of other transcription factors (including ATF-1, ATF-2, and CREB) known to bind CRE motifs. Na expression in cells results in increased levels of a hyperphosphorylated form of c-Jun, suggesting a mechanism by which Na activates c-Jun. Our results indicate that Na is a transcription factor that activates the EBV Zp IE promoter through its effects on c-Jun and suggest that Na cooperates with BRLF1 to induce the lytic form of EBV infection in certain cell types.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Diagram of the IE region of the EBV genome. The IE locus of EBV is depicted, with the positions of the BZLF1, BRLF1, BRRF1 (Na), and RAZ genes indicated. Promoters for BZLF1 (Zp), BRLF1 (Rp), and BRRF1 (Nap) are designated by arrows. Transcripts originating from each of the various promoters are depicted, and the proteins encoded (Z, R, RAZ, or Na) by each transcript are indicated above their respective mRNAs. The insertional mutations carried by the Z-KO virus and the R-KO virus are indicated by the gray boxes in the bottom diagram; numbers represent coordinates of the EBV strain B95.8 genome (accession no. V01555).
FIG. 2.
FIG. 2.
293 R-KO cells cannot transcribe BRRF1 mRNA. 293 WT, 293 Z-KO, or 293 R-KO cells were transfected with pSG5 (vector), pSG5-Z (Z), or pSG5-R (R). At 24 h posttransfection, total RNAs were harvested and Northern blotted as described in Materials and Methods. Membranes were hybridized with probes specific for BRRF1 (Na), R, Z, and GAPDH transcripts, as indicated by arrows.
FIG. 3.
FIG. 3.
Na enhances R-induced lytic infection in 293 R-KO cells. (A) 293 R-KO or 293 WT cells were transfected with pSG5 (vector) or pSG5-R (R). Cells were harvested at 48 h posttransfection, and 35 μg of protein was immunoblotted with antibodies specific for the early lytic protein BMRF1 (upper panel) or R (lower panel). (B) 293 R-KO cells were transfected with pSG5 (vector), pRC-FLAG-BRRF1 (Na), pSG5-R (R), or pRC-FLAG-BRRF1 and pSG5-R (Na+R). Cells were harvested at 48 h posttransfection, and 35 μg of protein was immunoblotted with antibodies specific for BMRF1, Z, R, FLAG (to detect FLAG-tagged Na), and β-actin, as indicated by arrows.
FIG. 4.
FIG. 4.
Na enhances R-induced lytic infection in a cell-line-dependent manner. AGS R-KO (A), BL30 R-KO (B), or LCL R-KO (C) cells were transfected with pRC (vector), pRC-FLAG-BRRF1 (Na), pSG5-R (R), or pRC-FLAG-BRRF1 and pSG5-R (Na+R). Cells were harvested at 48 h posttransfection, and 35 μg of protein was immunoblotted with the indicated antibodies. Densitometric analysis was performed with NIH Image software. AGS(−), EBV-negative AGS cells.
FIG. 5.
FIG. 5.
Na increases the ability of R to activate EBV replication in 293 R-KO cells. Supernatants from the cells transfected for Fig. 3B were harvested, and the amounts of infectious virus present were quantitated as described in Materials and Methods. x axis labels represent the transfected plasmids and y axis labels represent the numbers of infectious units per milliliter of supernatant harvested. The data shown represent the results of two independent experiments.
FIG. 6.
FIG. 6.
Na activates the Z promoter (Zp) in EBV-negative cells. HeLa cells were transfected with one of the indicated reporter constructs (Zp-CAT, −65Zp-CAT, EA-pBS-CAT, or pBS-CAT) in conjunction with either pRC (vector) or pRC-FLAG-BRRF1 (Na). Cells were harvested at 48 h posttransfection and CAT assays were performed. The data represent the results of two independent experiments.
FIG. 7.
FIG. 7.
Na activates Zp through a CRE site. (A) Diagram of the Zp reporter constructs used. The primary elements that positively regulate Zp are denoted in boxes, with the known transcription factors that bind them represented above. Site-directed mutations in either ZIA and ZIB or ZII are indicated with an “X.” (B) HeLa cells were transfected with one of the indicated reporter constructs (Zp-CAT, Zp-CAT ΔZIA/ZIB, Zp-CAT ΔZII) in conjunction with either pSG5 (vector) or pRC-FLAG-BRRF1 (Na). Cells were harvested at 48 h posttransfection and CAT assays were performed. The data represent the results of two independent experiments. (C) HeLa cells were transfected with one of the indicated reporter constructs [3CRE-CAT or 3CRE(mut)-CAT] in conjunction with either pRC (vector) or pRC-FLAG-BRRF1 (Na). Cells were harvested at 48 h posttransfection and CAT assays were performed. The data shown represent the results of two independent experiments.
FIG. 8.
FIG. 8.
Na activates c-Jun transactivator function. HeLa cells were transfected with Gal4-EIB-CAT in conjunction with a vector expressing one of the Gal4-fusion proteins (Gal4-ATF1, Gal4-ATF2, Gal4-CREB, or Gal4-c-Jun) or the control vector expressing Gal4 alone (Gal4). In addition, either pRC-FLAG-BRRF1 (Na) or pRC (vector) was also transfected into the cells. Cells were harvested and assayed for CAT activity at 48 h posttransfection. The data shown represent the results of two independent experiments.
FIG. 9.
FIG. 9.
Na increases levels of hyperphosphorylated c-Jun. HeLa cells were transfected with pRC (vector), pRC-FLAG-BRRF1 (Na), pCMV-c-Jun (c-Jun), or pRC-FLAG-BRRF1 plus pCMV-c-Jun (Na+c-Jun). Harvesting occurred at 48 h posttransfection, and 35 μg of protein was immunoblotted for c-Jun phosphorylated on Ser73 (P-c-Jun) (upper panel) or total c-Jun (lower panel).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adamson, A. L., D. Darr, E. Holley-Guthrie, R. A. Johnson, A. Mauser, J. Swenson, and S. Kenney. 2000. Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c-Jun N-terminal kinases. J. Virol. 74:1224-1233. - PMC - PubMed
    1. Adamson, A. L., and S. C. Kenney. 1998. Rescue of the Epstein-Barr virus BZLF1 mutant, Z(S186A), early gene activation defect by the BRLF1 gene product. Virology 251:187-197. - PubMed
    1. Bais, C., B. Santomasso, O. Coso, L. Arvanitakis, E. G. Raaka, J. S. Gutkind, A. S. Asch, E. Cesarman, M. C. Gershengorn, E. A. Mesri, and M. C. Gerhengorn. 1998. G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. Nature 391:86-89. - PubMed
    1. Binne, U. K., W. Amon, and P. J. Farrell. 2002. Promoter sequences required for reactivation of Epstein-Barr virus from latency. J. Virol. 76:10282-10289. - PMC - PubMed
    1. Borras, A. M., J. L. Strominger, and S. H. Speck. 1996. Characterization of the ZI domains in the Epstein-Barr virus BZLF1 gene promoter: role in phorbol ester induction. J. Virol. 70:3894-3901. - PMC - PubMed

Publication types