Epstein-Barr virus lytic infection is required for efficient production of the angiogenesis factor vascular endothelial growth factor in lymphoblastoid cell lines

Abstract

Although Epstein-Barr virus (EBV)-associated malignancies are primarily composed of cells with one of the latent forms of EBV infection, a small subset of tumor cells containing the lytic form of infection is often observed. Whether the rare lytically infected tumor cells contribute to the growth of the latently infected tumor cells is unclear. Here we have investigated whether the lytically infected subset of early-passage lymphoblastoid cell lines (LCLs) could potentially contribute to tumor growth through the production of angiogenesis factors. We demonstrate that supernatants from early-passage LCLs infected with BZLF1-deleted virus (Z-KO LCLs) are highly impaired in promoting endothelial cell tube formation in vitro compared to wild-type (WT) LCL supernatants. Furthermore, expression of the BZLF1 gene product in trans in Z-KO LCLs restored angiogenic capacity. The supernatants of Z-KO LCLs, as well as supernatants from LCLs derived with a BRLF1-deleted virus (R-KO LCLs), contained much less vascular endothelial growth factor (VEGF) in comparison to WT LCLs. BZLF1 gene expression in Z-KO LCLs restored the VEGF level in the supernatant. However, the cellular level of VEGF mRNA was similar in Z-KO, R-KO, and WT LCLs, suggesting that lytic infection may enhance VEGF translation or secretion. Interestingly, a portion of the vasculature in LCL tumors in SCID mice was derived from the human LCLs. These results suggest that lytically infected cells may contribute to the growth of EBV-associated malignancies by enhancing angiogenesis. In addition, as VEGF is a pleiotropic factor with effects other than angiogenesis, lytically induced VEGF secretion may potentially contribute to viral pathogenesis.

FIG. 1.

Z-KO LCL supernatants are less angiogenic than WT LCL supernatants in vitro. Supernatants from WT LCLs (WT) or Z-KO LCLs (Z-KO) growing in vitro were tested for their ability to induce tube or vessel formation of HDMECs in vitro. VEGF, HDMECs treated with the known angiogenic factor VEGF; (−) control, HDMECs treated with medium alone. Photomicrographs of a representative area of endothelial tube formation within a chamber for each sample from donor 1 and donor 2 are shown. Quantification of the total tube area per chamber formed by HDMECs treated with medium alone (control), medium from WT LCLs, or medium from Z-KO LCLs expressed as an angiogenic score (y axis) is shown for each donor. Data represent the means of three independent experiments, each done in triplicate. Error bars indicate standard errors of the mean. The results of LCLs derived from two different donors are shown.

FIG. 2.

Restoring the expression of BZLF1 in trans increases the angiogenic capacity of Z-KO LCLs. (a) Supernatants from Z-KO LCLs carrying a control vector (Z-KO-vector) or carrying a BZLF1 expression vector driven by the authentic BZLF1 promoter (Z-KO-ZpZ) were tested for their ability to induce tube and vessel formation of HDMECs in vitro. VEGF, HDMECs treated with the known angiogenic factor VEGF; (−) control, HDMECs treated with medium alone. Photomicrographs of a representative area of endothelial tube formation within a chamber for each sample are shown. (b) Quantification of the total tube area per chamber formed by HDMECs treated with medium alone (control), medium from Z-KO LCLs carrying a control vector (Z-KO-vector), or medium from Z-KO LCLs carrying a BZLF1 expression vector (Z-KO-ZpZ), expressed as an angiogenic score (y axis). Data represent the means of three independent experiments, each done in quadruplicate. Error bars indicate standard errors of the mean.

FIG. 3.

Examination of angiogenesis in vivo. Z-KO LCL tumors were stained using an antibody specific for human CD31. Examples of tumor vessels (a) and individual LCLs (b) staining positive for human CD31 are indicated by arrows. Similar results were obtained in WT LCL tumors (data not shown).

FIG. 4.

Lytic-defective LCLs secrete less VEGF than WT LCLs in vitro. (a) Supernatants from WT LCLs (WT), Z-KO LCLs (Z-KO), or R-KO LCLs (R-KO) growing in vitro were concentrated and immunoblotted using either VEGF-A- or bFGF-specific antibodies. Arrows indicate different forms of VEGF generated by alternative splicing. (b) Supernatants from Z-KO LCLs carrying a BZLF1 expression vector (Z-KO-ZpZ) and Z-KO LCLs carrying the control vector (Z-KO-vector) were concentrated and immunoblotted using a VEGF-A specific antibody.

FIG. 5.

Lytic infection does not regulate VEGF at the level of transcription. A comparison of VEGF (a) or B₂-microglobulin (B2-micro) (b) transcription using RT-PCR in WT, Z-KO, and R-KO LCLs growing in vitro is shown. VEGF transcripts are alternatively spliced, leading to multiple RT-PCR products. Different dilutions of starting cDNA amounts are shown to ensure linearity. A comparison of VEGF (c) or B₂-microglobulin (B2-micro) (d) transcription using RT-PCR in Z-KO LCLs carrying a control vector (Z-KO-vector) or a BZLF1 expression vector (Z-KO-ZpZ) is shown. Different dilutions of starting cDNA amounts are shown to ensure linearity.

FIG. 6.

Lytic-defective LCLs have a similar amount of intracellular VEGF as WT LCLs in vitro. (a) Cellular extracts from WT LCLs (WT), Z-KO LCLs (Z-KO), or R-KO LCLs (R-KO) growing in vitro were immunoblotted using the VEGF-A-specific antibodies. (b) Cellular extracts from Z-KO LCLs carrying a BZLF1 expression vector (Z-KO-ZpZ) and Z-KO LCLs carrying the control vector (Z-KO-vector) were immunoblotted using a VEGF-A-specific antibody.