The Epstein-Barr virus BamHI C promoter is not essential for B cell immortalization in vitro, but it greatly enhances B cell growth transformation.

UNLABELLED: Epstein-Barr virus (EBV) infection of B cells leads to the sequential activation of two viral promoters, Wp and Cp, resulting in the expression of six EBV nuclear antigens (EBNAs) and the viral Bcl2 homologue BHRF1. The viral transactivator EBNA2 is required for this switch from Wp to Cp usage during the initial stages of infection. EBNA2-dependent Cp transcription is mediated by the EBNA2 response element (E2RE), a region that contains at least two binding sites for cellular factors; one of these sites, CBF1, interacts with RBP-JK, which then recruits EBNA2 to the transcription initiation complex. Here we demonstrate that the B cell-specific transcription factor BSAP/Pax5 binds to a second site, CBF2, in the E2RE. Deletion of the E2RE in the context of a recombinant virus greatly diminished levels of Cp-initiated transcripts during the initial stages of infection but did not affect the levels of Wp-initiated transcripts or EBNA mRNAs. Consistent with this finding, viruses deleted for the E2RE were not markedly impaired in their ability to induce B cell transformation in vitro. In contrast, a larger deletion of the entire Cp region did reduce EBNA mRNA levels early after infection and subsequently almost completely ablated lymphoblastoid cell line (LCL) outgrowth. Notably, however, rare LCLs could be established following infection with Cp-deleted viruses, and these were indistinguishable from wild-type-derived LCLs in terms of steady-state EBV gene transcription. These data indicate that, unlike Wp, Cp is dispensable for the virus' growth-transforming activity. IMPORTANCE: Epstein-Barr virus (EBV), a B lymphotropic herpesvirus etiologically linked to several B cell malignancies, efficiently induces B cell proliferation leading to the outgrowth of lymphoblastoid cell lines (LCLs). The initial stages of this growth-transforming infection are characterized by the sequential activation of two viral promoters, Wp and Cp, both of which appear to be preferentially active in target B cells. In this work, we have investigated the importance of Cp activity in initiating B cell proliferation and maintaining LCL growth. Using recombinant viruses, we demonstrate that while Cp is not essential for LCL outgrowth in vitro, it enhances transformation efficiency by >100-fold. We also show that Cp, like Wp, interacts with the B cell-specific activator protein BSAP/Pax5. We suggest that EBV has evolved this two-promoter system to ensure efficient colonization of the host B cell system in vivo.

Epstein-Barr virus BamHI W repeat number limits EBNA2/EBNA-LP coexpression in newly infected B cells and the efficiency of B-cell transformation: a rationale for the multiple W repeats in wild-type virus strains.

The genome of Epstein-Barr virus (EBV), a gammaherpesvirus with potent B-cell growth-transforming ability, contains multiple copies of a 3-kb BamHI W repeat sequence; each repeat carries (i) a promoter (Wp) that initiates transformation by driving EBNA-LP and EBNA2 expression and (ii) the W1W2 exons encoding the functionally active repeat domain of EBNA-LP. The W repeat copy number of a virus therefore influences two potential determinants of its transforming ability: the number of available Wp copies and the maximum size of the encoded EBNA-LP. Here, using recombinant EBVs, we show that optimal B-cell transformation requires a minimum of 5 W repeats (5W); the levels of transforming ability fall progressively with viruses carrying 4, 3, and 2 W repeats, as do the levels of Wp-initiated transcripts expressed early postinfection (p.i.), while viruses with 1 copy of the wild-type W repeat (1W) and 0W are completely nontransforming. We therefore suggest that genetic analyses of EBV transforming function should ensure that wild-type and mutant strains have equal numbers (ideally at least 5) of W copies if the analysis is not to be compromised. Attempts to enhance the transforming function of low-W-copy-number viruses, via the activity of helper EBV strains or by gene repair, suggested that the critical defect is not related to EBNA-LP size but to the failure to achieve sufficiently strong coexpression of EBNA-LP and EBNA2 early postinfection. We further show by the results of ex vivo assays that EBV strains in the blood of infected individuals typically have a mean of 5 to 8 W copies, consistent with the view that evolution has selected for viruses with an optimal transforming function.

Epstein-Barr virus exploits BSAP/Pax5 to achieve the B-cell specificity of its growth-transforming program.

Epstein-Barr virus (EBV) can infect various cell types but limits its classical growth-transforming function to B lymphocytes, the cells in which it persists in vivo. Transformation initiates with the activation of Wp, a promoter present as tandemly repeated copies in the viral genome. Assays with short Wp reporter constructs have identified two promoter-activating regions, one of which (UAS2) appears to be lineage independent, while the other (UAS1) was B-cell specific and contained two putative binding sites for the B-cell-specific activator protein BSAP/Pax5. To address the physiologic relevance of these findings, we first used chromosome immunoprecipitation assays and found that BSAP is indeed bound to Wp sequences on the EBV genome in transformed cells. Thereafter, we constructed recombinant EBVs carrying two Wp copies, both wild type, with UAS1 or UAS2 deleted, or mutated in the BSAP binding sites. All the viruses delivered their genomes to the B-cell nucleus equally well. However, the BSAP binding mutant (and the virus with UAS1 deleted) showed no detectable activity in B cells, whether measured by early Wp transcription, expression of EBV latent proteins, or outgrowth of transformed cells. This was a B-cell-specific defect since, on entry into epithelial cells, an environment where Wp is not the latent promoter of choice, all the Wp mutant viruses initiated infection as efficiently as wild-type virus. We infer that EBV ensures the B-cell specificity of its growth-transforming function by exploiting BSAP/Pax5 as a lineage-specific activator of the transforming program.