Tierney Rosemary J, Nagra Jasdeep, Rowe Martin, Bell Andrew I, Rickinson Alan B
School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom
School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
J Virol. 2015 Mar;89(5):2483-93. doi: 10.1128/JVI.03300-14. Epub 2014 Dec 24.
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.
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.
EB病毒（EBV）感染B细胞会导致两个病毒启动子Wp和Cp的顺序激活，从而导致六种EB病毒核抗原（EBNAs）和病毒Bcl2同源物BHRF1的表达。在感染初始阶段，从使用Wp转换为使用Cp需要病毒反式激活因子EBNA2。EBNA2依赖的Cp转录由EBNA2反应元件（E2RE）介导，该区域包含至少两个细胞因子结合位点；其中一个位点CBF1与RBP-JK相互作用，然后将EBNA2招募到转录起始复合物中。在此，我们证明B细胞特异性转录因子BSAP/Pax5与E2RE中的第二个位点CBF2结合。在重组病毒背景下缺失E2RE会在感染初始阶段大幅降低Cp启动的转录本水平，但不影响Wp启动的转录本或EBNA mRNA的水平。与这一发现一致，缺失E2RE的病毒在体外诱导B细胞转化方面的能力并未明显受损。相比之下，整个Cp区域的更大缺失确实会在感染后早期降低EBNA mRNA水平，并随后几乎完全消除淋巴母细胞系（LCL）的生长。然而，值得注意的是，用缺失Cp的病毒感染后仍可建立罕见的LCL，并且这些LCL在稳态EBV基因转录方面与野生型来源的LCL没有区别。这些数据表明，与Wp不同，Cp对于病毒的生长转化活性是可有可无的。
EB病毒（EBV）是一种与多种B细胞恶性肿瘤病因相关的嗜B淋巴细胞疱疹病毒，能有效诱导B细胞增殖，导致淋巴母细胞系（LCL）生长。这种生长转化感染的初始阶段的特征是两个病毒启动子Wp和Cp的顺序激活，这两个启动子似乎在靶B细胞中优先活跃。在这项工作中，我们研究了Cp活性在启动B细胞增殖和维持LCL生长中的重要性。使用重组病毒，我们证明虽然Cp对于体外LCL生长不是必需的，但它可将转化效率提高100倍以上。我们还表明，Cp与Wp一样，与B细胞特异性激活蛋白BSAP/Pax5相互作用。我们认为EBV进化出这种双启动子系统以确保在体内有效地定殖于宿主B细胞系统。
