Mechanisms of Gene Regulation by EBV EBNA-1 Protein

描述(申请人提供)：爱泼斯坦-巴尔病毒(EBV)是一种非常成功的病原体，能够作为B淋巴细胞内的一种潜伏感染持续终生，几乎没有显性疾病。然而，免疫监测的崩溃，例如由于艾滋病，仍然是发展EBV相关淋巴瘤的一个重要风险因素，突显了这种潜在的致癌疱疹病毒和宿主免疫系统之间存在的高度进化的平衡。这种平衡依赖于在建立持续感染期间选择性地下调EBV潜伏期相关基因的表达，最终将表达限制在对维持持久性至关重要的病毒基因上，而排除那些具有急性转化特性和/或编码EBV特异性T细胞监测识别的优势表位的病毒基因。在这种向受限潜伏期转变的过程中，一个关键的过程是启动子切换事件，该事件使基本的EBV基因组维持蛋白EBNA-1能够从启动子QP排他性表达，而启动子QP可以通过紧靠其转录起始点下游的两个EBNA-1结合位点进行负向自动调节。我们最近对EBNA-1抑制机制的研究表明，它不是像最初认为的那样通过抑制转录发挥作用，而是通过抑制前mRNA的加工发挥作用。此外，这种自动监管的主要意义最近变得明显起来。尽管EBNA-1早期被认为对宿主免疫监视是“看不见的”，因为它有能力抑制细胞蛋白酶体对EBNA-1的降解，从而阻止EBNA-1与HLAI类分子相关的EBNA-1多肽表位的呈递，但随后的研究表明，识别EBNA-1的细胞毒性T细胞不仅存在，而且它们针对的是实际合成EBNA-1过程中产生的多肽，而不是成熟的EBNA-1的降解。因此，对蛋白酶体降解的抵抗力仅次于EBNA-1的自我调节表达，而EBNA-1是EBV用来限制EBNA-1特异性T细胞杀伤的主要机制。此外，最近描述的EBNA-1的抗凋亡特性表明，它可能具有致瘤潜力。因此，我们假设EBNA-1的自我调节功能对EBV的持久性及其相关的致病潜力非常关键：它确保足够的EBNA-1用于基因组维护，同时将EBNA-1的合成限制在一个阈值以下，如果超过这个阈值，将使潜伏感染的B细胞被EBNA-1特异性的细胞毒T细胞消除，并可能发生致癌转化。为了帮助我们实现确定EBNA-1自身调节对EBV生物学、免疫逃避和发病机制的贡献的长期目标，我们提出了三个具体目标：1)阐明EBNA-1对前mRNA加工的影响；2)确定EBNA-1自身调节的分子机制；以及3)阐明EBNA-1自身调节对EBV潜伏期的生长和限制性程序的贡献。公共卫生相关性：爱泼斯坦-巴尔病毒(EBV)是一种疱疹病毒，具有在其人类宿主中导致癌症的巨大潜力，例如，在因艾滋病而免疫抑制的个人中尤其如此。通过这项研究，我们希望更好地了解如何预防或治疗EBV感染。具体地说，我们试图阐明关键的EBV蛋白EBNA-1调节其自身表达的机制，并确定这种自我调节机制在B淋巴细胞内不同形式的EBV感染中的各自重要性。我们假设EBNA-1的自我调节功能确保了它的表达水平是在EBV DNA基因组的传播中发挥其基本作用所必需的，但低于宿主免疫系统可能检测到的可能促进恶性肿瘤的水平。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is an extremely successful pathogen, able to persist lifelong as a latent infection within B lymphocytes with little overt disease. However, a breakdown in immune surveillance, e.g., as a consequence of AIDS, remains a significant risk factor for development of EBV-associated lymphoma, underscoring the highly evolved equilibrium that exists between this potentially oncogenic herpesvirus and the host immune system. This equilibrium is dependent on a selective down-regulation of EBV latency-associated gene expression during establishment of persistent infection that ultimately restricts expression to viral genes critical for maintenance of persistence, while precluding those with acute transforming properties and/or which encode dominant epitopes recognized by the EBV-specific T-cell surveillance. A pivotal process in this transition to restricted latency is a promoter switching event that enables exclusive expression of the essential EBV genome-maintenance protein, EBNA-1, from the promoter Qp, which can be negatively autoregulated through two EBNA-1 binding sites immediately downstream of its transcription start site. Our recent efforts to define the mechanism of EBNA-1 repression revealed that it acts not by inhibition of transcription, as originally believed, but by suppression of pre-mRNA processing. The principal significance of this autoregulation, furthermore, has recently become apparent. Although EBNA-1 was earlier thought to be "invisible" to the host immune surveillance as a consequence of its ability to inhibit in cis its degradation by the cell proteasome, thereby preventing presentation of EBNA-1 peptide epitopes in association with HLA class I molecules, subsequent studies indicated that cytotoxic T cells that recognize EBNA-1 not only exist, but that they are directed towards peptides generated during actual synthesis of EBNA-1, not by the degradation of mature EBNA-1. Thus, resistance to proteasomal degradation is secondary to the autoregulated expression of EBNA-1 as the primary mechanism employed by EBV to restrict EBNA-1-specific T-cell killing. Further, recently described anti-apoptotic properties of EBNA-1 suggest that it may have tumorigenic potential. We hypothesize, therefore, that the autoregulatory function of EBNA-1 is highly critical to EBV persistence and its associated pathogenic potential: it ensures sufficient EBNA-1 for genome maintenance, while limiting EBNA-1 synthesis below a threshold that, if exceeded, would subject latently infected B cells to elimination by EBNA-1-specific cytotoxic T cells, and potentially oncogenic transformation. We propose three specific aims to help us reach our long-term objective of defining the contribution of EBNA-1 autoregulation to EBV biology, immune evasion and pathogenesis: 1) Elucidate the influence of EBNA-1 on pre-mRNA processing; 2) Define the molecular mechanism of EBNA-1 autoregulation; and 3) Elucidate the contributions of EBNA-1 autoregulation to the growth and restricted programs of EBV latency. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus (EBV) is a herpesvirus that has significant potential to cause cancer in its human host, particularly within individuals that become immune suppressed as a consequence of AIDS, for example. Through this research we hope to gain a better understanding of how infection by EBV may be prevented or treated. Specifically, we seek to elucidate the mechanism by which a key EBV protein, EBNA-1, regulates its own expression, and to determine the respective importance of this autoregulatory mechanism in the different forms of EBV infection within B lymphocytes. We hypothesize that the autoregulatory function of EBNA-1 ensures that it is expressed at levels necessary to perform its essential role in propagation of the EBV DNA genome, but below levels that would be detected by the host immune system and that might promote malignancy.