Lytic viral replication as a contributor to the detection of Epstein-Barr virus in breast cancer

Abstract

Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngeal carcinoma and has also been reported in other more controversial carcinoma settings. Evaluation of EBV association with epithelial carcinomas such as breast cancer would benefit from a better understanding of the outcome of EBV infection of these cells. Cell-free preparations of a green fluorescent protein-expressing virus, BX1, were used to infect breast cancer cell lines, which were then examined for EBV gene expression and viral genome copy number. Reverse transcription-PCR analyses revealed that the cells supported a mixture of latency II and lytic EBV gene expression. Lytic Zta and BMRF1 protein expression was detected by immunohistochemistry, and DNA PCR analyses estimated an EBV copy number of 300 to 600 genomes per infected cell. Evidence for lytic EBV expression was also found in breast tissue, where reverse transcription-PCR analyses detected lytic Zta transcripts in 7 of 10 breast carcinoma tissues and 4 of 10 normal tissues from the same patients. Scattered cells immunoreactive for Zta protein were also detectable in breast carcinoma. Quantitative real-time PCR analysis of EBV-positive breast carcinoma tissues suggested that less than 0.1% of the cells contained viral genomes. We suggest that sporadic lytic EBV infection may contribute to PCR-based detection of EBV in traditionally nonvirally associated epithelial malignancies.

FIG. 1.

In vitro EBV infection of breast cell lines. Breast cancer cell lines were incubated with concentrated, cell-free BX1 virus and monitored for GFP expression 48 h after infection. (A) MDA-MB231; (B) MDA-MB435; (C) MDA-MB468. Left panels, phase contrast. Right panels, immunofluorescence.

FIG. 2.

Latent and lytic EBV gene expression in infected MDA-MB468 cells. (A) Ethidium bromide-stained gels showing the DNA products amplified by nested RT-PCR with the specific primers listed in Table 1. mRNA was isolated from MDA-MB468 cells 48 h after infection with cell-free BX1 virus. The upper band present in the LMP1 RT-PCR migrates at the position expected for an unspliced LMP1 transcript. The minor bands in the Zta amplification represent partially spliced transcripts that retain either the first or second intron. Sizes indicated at the right were determined from the migration of φX174 DNA. (B) Diagram of the Zta coding region, showing the relative sizes (in base pairs) of the three exons and two introns and the locations of the primer (arrowheads) used in the RT-PCR analyses.

FIG. 3.

Detection of lytic Zta and BMRF1 protein expression in in vitro-infected MDA-MB468 cells. (A and B) Immunoperoxidase staining of MDA-MB468 cells fixed 48 h after infection with cell-free BX1 virus. (A) Anti-Zta primary antibody. (B) Anti-BMRF1 (EA-D) primary antibody. Positive-staining cells are indicated with arrows. (C and D) Immunofluorescence assay showing Zta expression in a converted MDA-MB468-BX1 cell line established by BX1 infection followed by selection for neomycin resistance. (C) Cells were stained with anti-Zta primary antibody and rhodamine-conjugated secondary antibody. (D) 4′,6′-Diamidino-2-phenylindole (DAPI) staining of cell nuclei. (E and F) MDA-MB468-BX1 cells triple stained for LMP1 expression (indocarbocyanine, orange), nuclear Zta expression (rhodamine, red), and DAPI (nuclear, blue) (E) or stained with DAPI alone (F).

FIG. 4.

Estimation of EBV genome copy number in MDA-MB468 cells infected in vitro. Real-time quantitative PCR was performed in duplicate with primers specific for the EBV BamHI-W fragment. (A) Amplification plots of the fluorescence intensity against PCR cycle number are shown to compare the EBV genome load in MDA-MB468 and virus-negative Akata (−) B cells 48 h after infection with cell-free BX1 virus. (B) EBV genome load in uninfected and infected MDA-MB468 cells versus EBV-positive Raji B cells. x axis, PCR cycle number. y axis, fluorescence intensity.

FIG. 5.

RT-PCR detection of EBV Zta transcripts in breast carcinoma and normal breast tissues. Ethidium bromide-stained gels showing the DNA products amplified by nested RT-PCR with the EBV Zta and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers described in Table 1. (A) Ten snap-frozen breast carcinoma samples. (B) Ten snap-frozen samples of normal breast tissue from the same patients. The major (closed arrow) and minor (open arrow) spliced forms of Zta are indicated. GAPDH amplification was used as a control for RNA integrity. Sizes indicated at the right were determined from the migration of φX174 DNA.

FIG. 6.

Comparison of the EBV genome load in EBV-positive breast carcinoma and normal breast tissues. Real-time quantitative PCR was performed with primers specific for the EBV BamHI-W fragment. Duplicate analysis of (A) five breast carcinoma samples (T2, T4, T5, T6, and T7) and (B) three normal breast samples (N2, N4, and N5) that were positive by RT-PCR for Zta expression. x axis, PCR cycle number. y axis, fluorescence intensity.

FIG. 7.

Detection of Zta protein expression in breast carcinoma tissue. Immunohistochemical staining of paraffin-embedded breast carcinoma tissue with anti-Zta primary antibody. Rare Zta-positive cells are indicated by arrows. Magnifications are indicated.