Noncoding RNAs in gamma-Herpesvirus Biology and AIDS Malignancies

总结 该项目的目的是使用KSHV、EBV和MHV 68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的RNA， 肿瘤发生我们的统一假设是A）γ-疱疹病毒利用短和长的非编码RNA， 调节病毒和宿主基因表达，和B）病毒非编码RNA和/或病毒干扰的宿主lncRNA 直接导致艾滋病相关恶性肿瘤的发生。为了支持这一假设，我们的程序 在第一个供资期内取得了一些开创性的发现，包括： 所有三种病毒的miRNA靶向组; γ-疱疹病毒感染中宿主lncRNA失调的证明 细胞;在所有三种病毒中发现circRNA; EBV和MHV 68转录组的全球分辨率， 发现新的非编码转录本;证明非编码RNA是主要的病毒基因 在某些EBV+恶性肿瘤中可检测到的产物;以及首次证明了长- EBER 1非编码RNA。到目前为止，我们的第一个周期的结果已经在20项研究中报道 出版物和5篇综述文章。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Renne博士（佛罗里达大学，UF）领导，将机械地研究病毒 lncRNA和KSHV编码的miRNA如何诱导HIV感染背景下宿主lncRNA表达的改变- 与KSHV相关的恶性肿瘤项目2由弗莱明顿博士（杜兰大学）领导，将询问 EBV（和KSHV）miRNA簇通过“微处理器过载”抑制宿主miRNA成熟 HIV相关的EBV恶性肿瘤的背景。Tibbetts（UF）将研究 MHV 68 miRNA和短非编码RNA与EBV EBER在小鼠慢性感染中的作用 肿瘤发生系统组织良好的行政核心（核心A，领导人：Rolf Renne）将继续 保持对项目的监督和组织，包括生物统计咨询和严格遵守， 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：Erik弗莱明顿）将继续产生很大的影响， 开发创新算法和数据分析管道。重组病毒核心（核心C，领导者： Rolf Renne）在UF，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 肿瘤发生核心（核心D，领导人：斯科特蒂贝茨）在UF将支持大量的分析， EBV和KSHV相关肿瘤样本，并进行所有拟定的肿瘤发生研究。总之，这 该计划项目已经对该领域产生了持续的影响，将进一步提高我们对 病毒非编码RNA和病毒干扰的宿主lncRNA如何促进γ-疱疹病毒肿瘤发生， 目标是确定新的脆弱性进行治疗干预。

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.