Noncoding RNAs in gamma-Herpesvirus Biology and AIDS Malignancies

摘要 该程序的目标是使用 KSHV、EBV 和 MHV68 短非编码和长非编码的比较分析 RNA揭示了γ-疱疹病毒非编码RNA的保守功能和其靶向的调控节点 肿瘤发生。我们的统一假设是 A) γ-疱疹病毒利用短和长非编码 RNA 调节病毒和宿主基因表达，B) 病毒非编码 RNA 和/或病毒扰动的宿主 lncRNA 直接导致 HIV 相关恶性肿瘤的发生。为了支持这个假设，我们的程序有 在第一个资助期间取得了一些开创性的发现，包括： 确定高可信度 所有三种病毒的 miRNA 靶标组； γ-疱疹病毒感染中宿主 lncRNA 失调的证明 细胞；发现所有三种病毒的 circRNA； EBV 和 MHV68 转录组的全局分辨率 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品；并首次展示长期体内功能 研究了 EBV EBER1 非编码 RNA。迄今为止，我们第一个周期的结果已在 20 项研究中报告 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 Renne 博士（佛罗里达大学，佛罗里达大学）领导，将机械地研究病毒 lncRNA 以及 KSHV 编码的 miRNA 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，将探讨 EBV（和 KSHV）miRNA 簇通过“微处理器过载”抑制宿主 miRNA 成熟 HIV 相关 EBV 恶性肿瘤的背景。由 Tibbetts 博士（佛罗里达大学）领导的项目 3 将研究 MHV68 miRNA 和短非编码 RNA 以及 EBV EBER 在小鼠慢性感染和 肿瘤发生系统。组织良好的行政核心（核心 A，领导者：Rolf Renne）将继续 维持对计划的监督和组织，包括生物统计咨询和遵守严格性， 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 杜兰大学生物信息学核心（核心 B，领导者：Erik Flemington）将继续产生巨大影响 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： UF 的 Rolf Renne）支持了 60 多种重组病毒的生成和质量控制， 将通过实施基于 CRISPR-Cas 的技术继续创新。临床样本和 佛罗里达大学的肿瘤发生核心（核心 D，领导者：Scott Tibbetts）将支持对大量肿瘤的分析 EBV 和 KSHV 相关肿瘤样本并进行所有提议的肿瘤发生研究。综上所述，这 计划项目已经对该领域产生了持续影响，将进一步加深我们对 病毒非编码RNA和病毒扰动的宿主lncRNA如何促进γ-疱疹病毒肿瘤发生 确定治疗干预的新脆弱性的目标。

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.