Cell-to-cell contact as an efficient mode of Epstein-Barr virus infection of diverse human epithelial cells

Abstract

We show clear evidence for direct infection of various human epithelial cells by Epstein-Barr virus (EBV) in vitro. The successful infection was achieved by using recombinant EBV (Akata strain) carrying a selective marker gene but without any other artificial operations, such as introduction of the known EBV receptor (CD21) gene or addition of polymeric immunoglobulin A against viral gp350 in culture. Of 21 human epithelial cell lines examined, 18 became infected by EBV, as ascertained by the detection of EBV-determined nuclear antigen (EBNA) 1 expression in the early period after virus exposure, and the following selection culture easily yielded a number of EBV-infected clones from 15 cell lines. None of the human fibroblasts and five nonhuman-derived cell lines examined was susceptible to the infection. By comparison, cocultivation with virus producers showed approximately 800-fold-higher efficiency of infection than cell-free infection did, suggesting the significance of direct cell-to-cell contact as a mode of virus spread in vivo. Most of the epithelial cell lines infectable with EBV were negative for CD21 expression at the protein and mRNA levels. The majority of EBV-infected clones established from each cell line invariably expressed EBNA1, EBV-encoded small RNAs, rightward transcripts from the BamHI-A region of the virus genome, and latent membrane protein (LMP) 2A, but not the other EBNAs or LMP1. This restricted form of latent viral gene expression, which is a central issue for understanding epithelial oncogenesis by EBV, resembled that seen in EBV-associated gastric carcinoma and LMP1-negative nasopharyngeal carcinoma. The results indicate that direct infection of epithelial cells by EBV may occur naturally in vivo, and this could be mediated by an unidentified, epithelium-specific binding receptor for EBV. The EBV convertants are viewed, at least in terms of viral gene expression, as in vitro analogs of EBV-associated epithelial tumor cells, thus facilitating analysis of an oncogenic role(s) for EBV in epithelial cells.

FIG. 1

Dual immunofluorescent staining of EBNA1 (green fluorescence) and cytokeratins (red fluorescence). Staining of a representative G418-resistant clone which appeared in a NU-GC-3 culture is shown. Magnification, ×400.

FIG. 2

Southern blot analysis of G418-resistant epithelial cells. The blots were probed with a BamHI-K fragment of EBV DNA. Serially diluted samples of Raji cell DNA (2.5, 1.25, 0.63, and 0.31 μg) served as positive controls. Each of the other pairs of lanes contained 5 μg of DNA extracted from the indicated EBV-negative parent epithelial cell lines and from their G418-resistant clones (left and right lanes, respectively, of each pair). All DNA samples were digested with BamHI.

FIG. 3

In situ detection of EBER1 expression in rEBV-infected epithelial cell lines. rEBV-converted NU-GC-3, RERF-LC-MS, and EBC-1 clones are shown in the top row, and their parental cells are shown in the bottom row. Strong nuclear signals can be seen in the EBV convertants but not in their parental cells. Magnification, ×600.

FIG. 4

Immunoblot analysis of EBV latent gene expression in virus-infected epithelial cells. (A) Expression of EBNAs and LMP1. Protein blots were probed with a pool of EBV-seropositive human sera for EBNAs and with CS1-4 MAbs for LMP1. Lysates extracted from 10⁵ cells of each rEBV-infected clone were used per lane. Lane labels indicate infected clones: MKN1/EBV, for example, indicates an rEBV-infected MKN1 clone. (B) Analysis of the clonal difference in LMP1 expression. Representative results for rEBV-infected clones from NU-GC-3, RERF-LC-MS, and DLD-1 cells are shown. Only two clones (NU-GC-3 clone 6 and RERF-LC-MS clone 4) were positive for LMP1. LCL, B-lymphoblastoid cell lines immortalized with rEBV as a positive control; BJAB, EBV-negative B-cell control.

FIG. 5

RT-PCR analysis of EBV latent gene expression in virus-infected epithelial cells. LCL, B-lymphoblastoid cell lines immortalized with rEBV, was used as a positive control for detection of LMP2A, LMP2B, BARF0, and Cp- or Wp-initiated EBNA mRNAs. rEBV-infected Akata⁻ (tk⁺) cells were used as a positive control for detection of Qp-initiated EBNA mRNA. HeLa cells served as a negative control. Labels on the lanes are explained in the legend to Fig. 4.

FIG. 6

CD21 expression in epithelial cells. (A) Flow cytometric analysis. Results for three representative epithelial cell lines highly susceptible to EBV infection are shown. A CD21-positive clone of BJAB (an EBV-negative B-cell line) was used as a positive control. The solid and dotted lines indicate staining with a mixture of anti-CD21 MAbs (HB5a and OKB7) and isotype controls, respectively. The vertical axis denotes the number of cells counted, and the horizontal axis denotes fluorescence intensity (log scale). (B) RT-PCR analysis. Transcription of CD21-specific mRNA was examined in all epithelial cell lines from which EBV convertants were isolated. Each lane of Raji represents amplification of cDNAs generated from serially fourfold-diluted total RNA (250, 62.5, 15.6, 4.0, and 1.0 ng of total RNA from left to right) from Raji cells as positive controls. The other lanes contained amplified products of 250 ng of total RNA from each epithelial cell line used in the study.