Viral Long Noncoding RNA Functions in Epstein-Barr Virus Infection

描述（由申请方提供）：EB病毒（EBV）是一种非常成功的病原体，能够在其人类宿主的B淋巴细胞内建立终身潜伏感染，当感染延迟至生命的第二个十年时，健康个体的显性疾病仅限于约40%的个体的自限性单核细胞增多症。然而，细胞免疫的破坏，特别是作为AIDS的结果，仍然是EBV相关淋巴瘤和淋巴增生性疾病发展的重要风险因素，强调了EBV与其宿主之间存在的高度进化的平衡。这种平衡的建立和EBV的致癌潜力依赖于EBV潜伏期相关基因的高度协调和受控表达，所述基因不仅编码蛋白质，而且编码至少四类非编码RNA（ncRNA）。本申请中提出的研究的长期目标是了解属于长ncRNA（lncRNA）类别的两种EBV RNA对EBV感染和致病潜力的贡献，即，长度超过200个核苷酸的RNA，几乎没有或没有功能性蛋白质编码能力。这些lncRNA由旁系同源的EBV基因BHLF1和LF3编码，长期以来已知在病毒复制或裂解周期期间以高水平表达，但最近被鉴定为在潜伏感染期间也活跃表达。我们的初步结果表明，在从EBV基因组中删除BHLF 1位点后，该病毒不能维持EBV潜伏相关蛋白的完全表达，转化为与其细胞储库（记忆B细胞）内的长期EBV持久性相关的更受限制的潜伏基因表达程序。因此，我们假设BHLF1 lncRNA及其来自LF3的parcRNA在EBV潜伏感染和持续性的调节中发挥积极但不同的作用。我们提出四个具体目标：在目标1下，我们将鉴定与BHLF1 lncRNA相互作用的蛋白质，以及这些特异性RNA：蛋白质缔合在EBV潜伏感染中发挥的作用。在目标2中，我们将阐明BHLF1 lncRNA在不同EBV潜伏期程序（由病毒基因表达的特定模式定义）期间的表达模式，以及它们是否运输到具有已知功能的特定亚细胞结构域，可能揭示EBV lncRNA的功能。在目标3中，我们将阐明BHLF1基因位点本身是否与其lncRNA一起通过调节病毒基因组构型和其他机制作为EBV潜伏期程序的中心决定因素。最后，在目标4下，我们将开始定义LF3基因座和lncRNA对EBV潜伏期的作用，采取与我们对BHLF 1提出的类似的策略。

DESCRIPTION (provided by applicant): Epstein-Barr virus (EBV) is an extremely successful pathogen, being able to establish a lifelong latent infection within B lymphocytes of its human host, with overt disease in healthy individuals restricted to a self- limiting mononucleosis in ~40% of individuals when infection is delayed until the second decade of life. However, a breakdown in cellular immunity, particularly as a consequence of AIDS, remains a significant risk factor for development of EBV-associated lymphoma and lymphoproliferative disease, underscoring the highly evolved equilibrium that exists between EBV and its host. Establishment of this equilibrium, and EBV's oncogenic potential are dependent on a highly coordinated and controlled expression of the EBV latency- associated genes, which encode not only proteins but at least four classes of noncoding RNAs (ncRNAs). The long-term objective of the research proposed in this application is to understand the contributions made to EBV infection and pathogenic potential by two EBV RNAs belonging to the class of long ncRNAs (lncRNAs), i.e., RNAs longer than 200 nucleotides with little or no functional protein-encoding capability. These lncRNAs are encoded by the paralogous EBV genes BHLF1 and LF3, long known to be expressed at high level during the virus replication or lytic cycle, but recently identified as beig actively expressed during latent infection as well. Our preliminary results indicate that, upon deletion of the BHLF1 locus from the EBV genome, the virus is unable to sustain full expression of the EBV latency-associated proteins, converting to a more restricted program of latency-gene expression associated with long-term EBV persistence within its cellular reservoir, the memory B cell. Thus, we hypothesize that the BHLF1 lncRNA, and its paralog from LF3, play active but distinct roles in the regulation of EBV latent infection and persistence. We propose four specific aims: Under Aim 1, we will identify proteins that interact with the BHLF1 lncRNA, and the role that these specific RNA:protein associations play in EBV latent infection. In Aim 2 we will elucidate the expression patterns of the BHLF1 lncRNAs during the different EBV latency programs (defined by specific patterns of viral gene expression), and whether they traffic to specific subcellular domains with known functionality, possibly revealing function(s) of the EBV lncRNA. In Aim 3 we will elucidate whether the BHLF1 gene locus itself, in concert with its lncRNA, acts as a central determinant of EBV latency program by regulating viral genome configuration and other mechanisms. Finally, under Aim 4 we will begin to define the roles that the LF3 locus and lncRNA contribute to EBV latency, taking a similar tack as we have proposed for BHLF1.