Secretion of Epstein-Barr virus-encoded BARF1 oncoprotein from latently infected B cells

Abstract

Epstein-Barr virus (EBV) encodes two oncogenes, LMP1(Latent Membrane Protein-1) and BARF1 (BamH1-A Reading Frame-1). LMP1 belongs to latent gene family and BARF1 is considered so far as one of early gene family. However BARF1 oncogene was expressed highly in Nasopharyngeal (NPC) and gastric (GC) carcinoma as a type II latency, and in EBV-positive Akata cell and primary epithelial cell infected in vitro by EBV as type I latency. Its expression was also reported in Burkitt's lymphoma's biopsy frequent in Malawi in Africa as well as in nasal NK/T-cell lymphoma. We recently observed a massive secretion of BARF1 protein in serum and saliva of NPC patients. NPC-derived c666-1 epithelial cells also expressed and secreted BARF1 protein without other lytic genes expression. We asked whether this oncogene belongs to latent gene family. To investigate, we examined its transcriptional and translational expression in IB4 and Akata B cells where both cell lines belong to latent cell family. Transcriptional expression was analyzed by RT-PCR. As BARF1 protein is one of secreted proteins, its translational expression was analyzed by immunoblot after concentration of culture medium. Secreted BARF1 protein was futher purified by concanavalin A affinity column. BARF1 was transcribed in both EBV-positive AKATA and IB4 cells, and BARF1 protein was secreted from these latently infected human B cells. Its secretion does not depend EBV genome form in infected cells. Both episomal and integrated form of EBV genome were capable of expressing BARF1 gene. These results suggests that BARF1 is expressed in latent stage and increases its expression during lytic stage.

Figure 1

Transcriptional and translational expression of BARF1 in latently infected AKATA and IB4 cells. A. Transcriptional expression of BARF1 on EBV-positive cell lines and BARF1 negative Raji cell line by RT-PCR. mRNA was purified using bead polyA extraction column (Promega, France). Five μg of mRNA was used for first-strand cDNA synthesis using oligo(dT)₁₅ as primer. Reverse transcription was done with Superscript reverse transcriptase (GIBCO, BRL). Amplifications of cDNA were performed in a DNA thermal cycler using the previously described primers (12). Amplified fragment was electrophoresed on 2% agarose gel, then transfered onto nitrocellulose. RT+: with reverse transcriptase. RT-: PCR directly with RNA without reverse transcription. Amplified actin sequence was presented as Actin. B. Radioactive hybridization. The hybridization was carried out in 6 × SSC, 0.5% SDS, 3 × Denhart and 200 μg/ml denatured salmon sperm DNA [8], with 10⁶ cpm/ml of labelled probe [25]. The filter was exposed for 2 hours at -80°C, then developed. C. Presence of p29 BARF1 protein in culture medium of EBV-positive cell line (IB4 and AKATA) and BARF1-negative Raji cell line. To purify secreted BARF1 protein, the concentrated medium was incubated with concanavalin A-ag at room temperature, then concanavalin A-ag was washed and elution of the conA-bound proteins was carried out by competition with methyl-ζ-D-glucopyranocide (MGP, 0.5–1.0 M; Sigma) as already described (20). 29 kDa corresponds to M.W of purified BARF1 protein. 25 kDa cprresponds to light chain of immunoglobuline.