Noncoding RNAs in gamma-Herpesvirus Biology and AIDS Malignancies

摘要 本程序的目标是使用KSHV、EBV和MHV68的短和长非编码的比较分析 揭示γ疱疹病毒非编码RNA的保守功能和调控节点 肿瘤发生学。我们的统一假设是：A)γ疱疹病毒利用长短的非编码RNA 调节病毒和宿主基因的表达，以及B)病毒非编码RNA和/或病毒干扰的宿主LncRNA 与艾滋病毒相关的恶性肿瘤的发生直接相关。为了支持这一假设，我们的节目已经 在第一个供资期间取得了一些开创性的发现，包括：确定高度信任 三种病毒中的miRNA靶标；γ疱疹病毒感染中宿主lncRNA失控的证明 细胞；在所有三种病毒中发现CircRNA；EBV和MHV68转录本的全球分辨率以及 发现新的非编码转录本；证明非编码RNA是主要的病毒基因 在一些EBV恶性肿瘤中可检测到的产品；并展示了第一个体内功能的长期- 研究了EBV EBER1非编码RNA。到目前为止，我们第一个周期的结果已经在20项研究中得到了报道。 出版物和5篇评论文章。为了继续解决我们的统一假设，我们提出了三个高度 综合项目：项目1，由雷恩博士(佛罗里达大学，UF)领导，将机械地研究病毒 以及KSHV编码的miRNAs如何在HIV背景下诱导宿主lncRNA表达的改变- 相关的KSHV恶性肿瘤。项目2，由弗莱明顿博士(杜兰大学)领导，将审问 EBV(和KSHV)miRNA簇通过“微处理器过载”抑制宿主miRNA成熟 与艾滋病毒相关的EBV恶性肿瘤的背景。由Tibbetts博士(UF)领导的项目3将研究 在易感的小鼠慢性感染和EB病毒感染中MHV68miRNAs和短非编码RNAs和EBVeber 肿瘤发生系统。组织良好的行政核心(核心A，领导人：罗尔夫·雷恩)将继续 保持对计划的监督和组织，包括生物统计学咨询和坚持严谨， 再现性和透明度标准。三个服务核心支持所有三个项目：rna-seq和 杜兰大学的生物信息学核心(核心B，领导者：埃里克·弗莱明顿)将在 开发创新的算法和数据分析管道。重组病毒核心(核心C，领导者： 罗尔夫·雷恩)在UF，该公司支持了60多种重组病毒的产生和质量控制， 将继续通过实施基于CRISPR-CAS的技术进行创新。临床样本和 UF的肿瘤发生核心(核心D，领导者：Scott Tibbetts)将支持对大量 EBV和KSHV相关肿瘤样本，并执行所有拟议的肿瘤发生研究。总而言之，这是 已经在实地产生持续影响的方案项目将进一步增加我们对 病毒非编码RNA和受病毒干扰的宿主lncRNA如何促进γ-疱疹病毒肿瘤的发生 为治疗干预定义新的脆弱性的目标。

SUMMARY The goal of this program is to use comparative analysis of KSHV, EBV and MHV68 short and long noncoding RNAs to reveal conserved functions of, and regulatory nodes targeted by, γ-herpesvirus noncoding RNAs during tumorigenesis. Our unifying hypothesis is that A) γ-herpesviruses utilize short and long noncoding RNAs to regulate both virus and host gene expression, and B) viral noncoding RNAs and/or virus-perturbed host lncRNAs directly contribute to the genesis of HIV-associated malignancies. In support of this hypothesis, our program has made a number of seminal findings during the first funding period, including: identification of high confidence miRNA targetomes across all three viruses; demonstration of host lncRNA de-regulation in γ-herpesvirus infected cells; discovery of circRNAs across all three viruses; global resolution of EBV and MHV68 transcriptomes and discovery of new noncoding transcripts; demonstration that noncoding RNAs are the predominant viral gene products detectable in some EBV+ malignancies; and demonstration of the first in vivo function for the long- studied EBV EBER1 noncoding RNA. To date, our results from the first cycle have been reported in 20 research publications and 5 review articles. To continue to address our unifying hypothesis we propose three highly integrated projects: Project 1, led by Dr. Renne (University of Florida, UF), will mechanistically study viral lncRNAs and how KSHV-encoded miRNAs induce alterations of host lncRNA expression in the context of HIV- associated KSHV malignancies. Project 2, led by Dr. Flemington (Tulane University) will interrogate the role of EBV (and KSHV) miRNA cluster inhibition of host miRNA maturation through “microprocessor overload” in the context of HIV-associated EBV malignancies. Project 3 led by Dr. Tibbetts (UF) will investigate the function of MHV68 miRNAs and short noncoding RNAs and EBV EBERs in a facile murine chronic infection and tumorigenesis system. The well-organized Administrative core (Core A, Leader: Rolf Renne) will continue to maintain oversight and organization of the program, including biostatistical consultation and adherence to rigor, reproducibility, and transparency standards. Three service cores support all three projects: The RNA-seq and Bioinformatics Core (Core B, Leader: Erik Flemington) at Tulane University will continue to have high impact by developing innovative algorithms and data analyses pipelines. The Recombinant Virus Core (Core C, Leader: Rolf Renne) at UF, which has supported the generation and quality control of more than 60 recombinant viruses, will continue to innovate by implementing CRISPR-Cas-based techniques. The Clinical Sample and Tumorigenesis Core (Core D, Leader: Scott Tibbetts) at UF will support the analysis of a significant numbers of EBV and KSHV-associated tumor samples and perform all proposed tumorigenesis studies. In summary, this program project, which has already had sustained impact on the field, will further increase our understanding of how viral noncoding RNAs and virus-perturbed host lncRNAs contribute to γ-herpesvirus tumorigenesis with the goal of defining new vulnerabilities for therapeutic intervention.