Epstein-Barr Virus (EBV)-Encoded RNA 2 (EBER2) but Not EBER1 Plays a Critical Role in EBV-Induced B-Cell Growth Transformation (cid:1)

Epstein-Barr virus (EBV)-encoded RNA 1 (EBER1) and EBER2 are untranslated RNAs and the most abundant viral transcripts in latently EBV-infected cells. We previously reported that EBERs play a critical role in efﬁcient EBV-induced growth transformation of primary B cells. To investigate whether EBER1 and EBER2 have distinct roles in B-cell growth transformation, recombinant EBVs carrying either EBER1 or EBER2 were generated. The transforming ability of recombinant EBVs expressing EBER2 was as high as that of EBVs expressing both EBER1 and EBER2. In contrast, the transforming ability of recombinant EBVs carrying EBER1 was impaired and was similar to that of EBV lacking both EBER1 and EBER2. Lymphoblas- toid cell lines (LCLs) established with EBVs carrying EBER2 proliferated at low cell densities, while LCLs established with EBVs carrying EBER1 did not. Interleukin 6 (IL-6) production in LCLs expressing EBER2 was more abundant than in those lacking EBER2. The growth of LCLs lacking EBER2 was enhanced by the addition of recombinant IL-6 to the cell culture, while the growth of EBER2-expressing LCLs was inhibited by a neutralizing anti-IL-6 antibody. These results demonstrate that EBER2, but not EBER1, contributes to efﬁcient B-cell growth transformation. We conclude that EBER1 and EBER2, despite their structural simi- larity, have different functions in latently infected lymphoblastoid cells. (cid:4) (cid:4) hygromycin resistance gene the simian cloned into the BamHI site of (Life nologies), hygromycin resistance loxP NotI (cid:4) pUC-A/C-PstI/EcoK (cid:4) pEBER1-KI pEBER2-KI, pEBER1-KI or pEBER2-KI was used as a tar- geting construct to generate EBER1-knockin EBV or EBER2-knockin EBV, Other known EBV open reading frames were not affected by this insertion. pSG-Cre was constructed by cloning the blunted XhoI-MluI fragment of pBS185 (Life Technologies), containing the cre recombinase gene, into the blunted BglII site of pSG5 (Stratagene). tions. To detect the expression of EBV latent genes, cDNA synthesis was per- formed using Moloney murine leukemia virus reverse transcriptase (MMLV RT [Invitrogen]) and 100 pmol of random primer (Takara) at 37°C for 60 min, followed by heat inactivation at 94°C for 10 min. The cDNA samples were then subjected to 30 cycles of PCR. The PCR products were electrophoresed on an agarose gel and transferred to a Hybond N (cid:1) membrane, and ampliﬁed DNA was detected using the Gene Images 3 (cid:6) -oligolabeling module (Amersham) and ECL detection reagents. The quality of the isolated RNA was veriﬁed by PCR ampliﬁcation of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. To detect the expression of cytokine genes, cDNA synthesis was performed using MMLV RT and an oligo(dT) primer (Promega), and then the cDNA samples were subjected to PCR as follows. For the detection of tumor necrosis factor alpha (TNF- (cid:2) ) expression, primers 5 (cid:6) -CTTCTGCCTGCTGCACTTTGG A-3 (cid:6) and 5 (cid:6) -TCCCAAAGTAGACCTGCCCAGA-3 (cid:6) were used and the PCR conditions were 25 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. For the detection of TNF- (cid:7) expression, primers 5 (cid:6) -AAGCTGCCAGAGGAGGAG CC-3 (cid:6) and 5 (cid:6) -TCCCGCTCGTCAGAAACGCC-3 (cid:6) were used, and the PCR con- ditions were 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. For the detection of IL-6 expression, primers 5 (cid:6) -TACATCCTCGACGGCATCTC-3 (cid:6) and 5 (cid:6)