"Project 2" Microprocessor overload in gamma-herpesviral oncogenesis

总结 爱泼斯坦巴尔病毒（EBV）是霍奇金淋巴瘤、非霍奇金淋巴瘤、胃淋巴瘤、胃淋巴瘤、淋巴瘤 癌症、鼻咽癌和自身免疫性疾病。在艾滋病毒合并感染（+或- ART）的情况下， EBV是特别成问题的，具有EBV相关淋巴瘤的显著升高的风险。整体 本P01的目的是研究非编码RNA在HIV相关恶性肿瘤中的作用。我们 对EBV相关伯基特淋巴瘤（BL）和胃癌（GC）患者数据集的评估显示， 除了广泛表达的EBER 1/2非编码小病毒RNA外，体内病毒基因表达是 主要限于两个重叠的长非编码RNA，A73和RPMS 1和两个大的miRNA簇 包含在RPMS 1内含子中。引人注目的是，我们发现病毒miRNAs占所有miRNAs的75 在BL和GC临床样品中的细胞中表达。miRNA targetome分析显示，EBV（和 KSHV（项目1））miRNAs与其靶点的相互作用比其细胞对应物更有效，部分原因是 通过靶向RNA更易接近的区域， 能量在功能上，我们发现EBV miRNAs可能抑制先天性和适应性免疫反应， 病毒感染总之，这些研究支持了以下论点，即EBV miRNAs是导致EBV感染的关键因素。 EBV相关淋巴瘤的肿瘤表型。 虽然某些细胞miRNAs是已知的致癌基因，但有广泛的证据表明， 癌细胞中miRNAs的表达。此外，miRNA加工基因中的驱动突变已经被发现。 在许多癌症类型中被发现。利用患者RNA-seq数据，我们发现细胞miRNA加工是 在EBV阳性的肿瘤中减少，我们发现EBV miRNA簇在EBV阴性的细胞中表达， 导致内源性细胞miRNA的加工减少。我们假设， 在EBV感染后，异位高密度miRNA簇的拷贝进入细胞不仅产生高水平的病毒miRNA， 而且还导致微处理器资源的隔离和细胞的抑制处理 miRNAs。我们进一步假设病毒微处理器抑制减少了细胞肿瘤的表达， 抑制性miRNA以类似于miRNA的遗传改变的方式影响肿瘤表型 加工因子基因 在本提案中，我们将在组织中使用实验室工作、病毒重组工程（核心C）和生物信息学（核心B 培养、动物模型（核心D）和临床模型，以1）确定直接病毒miRNA的功能影响 靶向免疫调节途径和2）描绘潜在的机制，影响 致癌途径，以及EBV介导的微处理器过载对肿瘤的功能后果 表型。我们还将开始初步研究，以确定微处理器过载是否是一个共享的 通过测试KSHV（项目1）miRNA簇，将特征扩展到其他致癌疱疹病毒。

Summary The Epstein Barr virus (EBV) is a causal agent in Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, gastric cancer, nasopharyngeal carcinoma and autoimmune diseases. In the context of HIV co-infection (+ or - ART), EBV is especially problematic with a substantially elevated risk of EBV-associated lymphomas. The overall objective of this P01 is to investigate the role of non-coding RNAs in HIV-associated malignancies. Our assessment of EBV-associated Burkitt’s lymphoma (BL) and gastric cancer (GC) patient datasets revealed that outside of the ubiquitously expressed small EBER1/2 non-coding viral RNAs, in vivo viral gene expression is primarily limited to two overlapping long non-coding RNAs, A73 and RPMS1 and two large miRNA clusters contained within the RPMS1 introns. Strikingly, we found that viral miRNAs comprise up to 75% of all miRNAs expressed in the cell in both BL and GC clinical samples. MiRNA targetome analyses revealed that EBV (and KSHV (Project 1)) miRNAs interact more effectively with their targets than their cellular counterparts, in part through targeting more accessible regions of RNAs and through forming hybrids with more favorable binding energies. Functionally, we found that EBV miRNAs likely suppress innate and adaptive immune responses to viral infection. Together, these studies support the contention that EBV miRNAs are key contributors to the tumor phenotype in EBV associated lymphomas. Although certain cell miRNAs are known oncogenes, there is extensive evidence for a generalized lower expression of cell miRNAs in cancer. Further, driver mutations in miRNA processing genes have been identified in a number of cancer types. Utilizing patient RNA-seq data, we found that cell miRNA processing is diminished in EBV positive tumors and we show that expression of EBV miRNA clusters in EBV negative cells causes decreased processing of endogenous cell miRNAs. We hypothesize that the introduction of multiple copies of ectopic high-density miRNA clusters into cells upon EBV infection not only yields high viral miRNA production but also causes sequestration of microprocessor resources and inhibited processing of cell miRNAs. We further hypothesize that viral microprocessor inhibition diminishes the expression of cell tumor suppressor miRNAs to influence the tumor phenotype in a manner similar to genetic alterations of miRNA processing factor genes. In this proposal, we will use bench work, viral recombineering (Core C), and bioinformatics (Core B) in tissue culture, animal models (Core D), and clinical models to 1) determine the functional impact of direct viral miRNA targeting on immune regulatory pathways and 2) delineate the underlying mechanisms, the impacted oncogenic pathways, and the functional consequence of EBV-mediated microprocessor overload on the tumor phenotype. We will also begin preliminary studies to determine whether microprocessor overload is a shared feature extending to other oncogenic herpesviruses through testing the KSHV (Project 1) miRNA cluster.