The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Abstract

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.

FIG 1

Identification of BDLF4 protein. (A) B95-8 cell lysate, BDLF4 protein synthesized in vitro, and BDLF4 expressed in HeLa cells were subjected to Western blotting. B95-8 cells were incubated with TPA (200 ng/ml), A23187 (0.5 μM), and sodium butyrate (5 mM) (T/A/B) to induce the lytic cycle. (B) Expression levels of BDLF4, BZLF1, BALF5 (Pol), and BALF4 (gB) in B95-8 cells were assessed by Western blotting at the indicated hours after lytic induction. (C) B95-8 cells were treated with PAA to examine the kinetics of BDLF4 expression. Lytic induction was carried out for 48 h (T/A/B) in the presence or absence of 400 μg/ml of PAA. Cells lysates were subjected to RT-PCR using BALF4 (gB), BDLF4, BALF2, and BZLF1 primers as described previously (24).

FIG 2

Construction of a point mutant of EBV BDLF4. (A) BDLF4 knockout EBV and its revertant virus were generated using a BAC system in Escherichia coli (25, 26). The neomycin resistance and streptomycin sensitivity genes (Neo/St) were inserted between nucleotides 220 (nt220) and nt221 of the BDLF4 gene. The Neo/St cassette was then replaced by a BDLF4 sequence with a one-nucleotide deletion at T (nt222) (dBDLF4-Frame). This one nucleotide excision caused frame shifting in the BDLF4 gene at residue 74 and creation of a new termination signal after synthesizing 12 nonsense, unrelated amino acids. The Neo/St cassette was again inserted and replaced with a wild-type BDLF4 sequence to make the revertant virus dBDLF4/R. (B) Electrophoresis of the recombinant viruses. EBV BAC DNAs were digested with BamHI or EcoRI and separated in an agarose gel.

FIG 3

DNA synthesis (A), progeny production (B), viral protein expression (C), and viral mRNA levels (D) in wild-type, BDLF4 mutant, and revertant viruses. (A) Viral DNA was prepared from HEK293 cells that carry the indicated recombinant EBV-BAC genomes at 0 or 72 h after transfection of the BZLF1 expression vector and was analyzed by quantitative real-time PCR. (B) Supernatants were collected at 72 h after transfection of pcDNA-BZLF1. Virus titers in 1 ml of supernatants were determined by examining the levels of green fluorescent protein (GFP) expression in Akata(-) cells after 2 days by fluorescence-activated cell sorter (FACS) analysis. (C) HEK293 cells were induced lytically by transfection of pcDNA-BZLF1. Whole-cell lysates were prepared at 0 and 48 h and analyzed by Western blotting using anti-BDLF4, anti-BALF2, anti-BMRF1, anti-BALF4 (gB), anti-BRRF2, anti-BZLF1, and anti-tubulin antibodies. (D) Real-time RT-PCR was carried out to determine expression of viral late genes (MCP [major capsid protein], gP350, and BALF4 [gB]) and an early gene. HEK293 cells carrying the indicated EBV genomes were transfected with pcDNA-BZLF1 and harvested after 72 h. As shown in this figure, two independent cell clones were tested for wild-type, dBDLF4-Frame, and dBDLF4/R. Bars represent the means and standard deviations of results from three independent experiments.

FIG 4

Loss of BDLF4 is responsible for the inhibition of late gene expression (A) HEK293 cells latently infected with EBV were transfected with the indicated expression vector and harvested after 96 h. After freeze-thawing and centrifugation, the supernatants were cultured with Akata(-) cells. FACS analysis was performed to count GFP-positive cells. (B) Levels of viral proteins. HEK293 cells with recombinant EBV were transfected with the indicated vector. Whole-cell lysates were prepared at 0 and 72 h and analyzed by Western blotting with anti-BALF2, anti-BDLF4, anti-BZLF1, anti-BALF4 (gB), and anti-tubulin. (C) Levels of viral late mRNAs. HEK293 cells with recombinant EBVs were transfected with the indicated vector. The mRNAs were prepared at 72 h and analyzed by real-time RT-PCR. (D) Association of BDLF4 with other late gene regulators. HEK293 cells were transfected with expression vectors for Flag-BDLF4, Myc-BGLF3, hemagglutinin-BcRF1 (HA-BcRF1), HA-BVLF1, and HA-BDLF3.5. Immunoprecipitation (IP) was carried out using anti-Flag antibody, and the results were detected by Western blotting using anti-Myc, anti-HA, and anti-Flag antibodies. Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a negative control. The arrowhead indicates immunoglobulin.