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.