Noncoding RNAs in gamma-Herpesvirus Biology and AIDS Malignancies

项目摘要 卡波西肉瘤相关疱疹病毒（KSHV）是一种人类γ-疱疹病毒，是艾滋病的病原体 恶性肿瘤如KS和原发性渗出性淋巴瘤（PEL）。近年来，很明显， 疱疹病毒包括EBV、KSHV和MHV 68表达许多长的非编码RNA（lncRNA）， 其与蛋白质编码转录物处于反义方向。这些RNA的功能和结构是 大部分未知。此外，这些病毒表达microRNA（miRNAs）。在描述KSHV的特征时， 使用改良的杂交体交联和测序（qCLASH）方案，我们鉴定了miRNA靶向组， 数百种宿主细胞lncRNA作为推定的miRNA靶。这些数据强烈表明，KSHV 编码的蛋白质和miRNA导致宿主lncRNA的失调。重要的是，34种lncRNA KSHV感染后的干扰，包括MALAT 1，HOTTIP，ANRIL，Meg 3，UCA 1和GAS-5 与人类癌症有关。我们还将mRNA和lncRNA靶点与癌症标志物 表型，如增殖，迁移，血管生成和葡萄糖代谢，并开始确定 作为KS模型的人内皮细胞中KSHV miRNA干扰的信号通路。我们也 将异常剪接鉴定为另外的癌症标志表型。在这里，我们建议扩展我们的研究， 通过整合来自qCLASH、RNAseq和miRNAseq数据的多组学数据集， KSHV感染的内皮细胞中miRNA调控的基因调控网络此外，我们建议验证 我们在大量人类肿瘤样本中的发现， qCLASH的targetome。这些研究将与项目2和项目3进行比较， 此外，还将得到核心B、C和D的支持。此外，我们还在功能上研究 反义拉娜转录本（ALT），我们已经使用高度特异性的PCR方法鉴定了81种推定的结合蛋白。 创新的RNA下拉分析。此外，我们将研究一类新发现的病毒环状RNA的作用， 被这个程序发现了。总之，这个项目的目标是描绘病毒的作用， 在AIDS恶性肿瘤中被病毒感染干扰的lncRNA和宿主细胞lncRNA。

PROJECT SUMMARY Kaposi's sarcoma-associated herpesvirus (KSHV), a human gamma-herpesvirus, is the causative agent of AIDS malignancies like KS and primary effusion lymphomas (PEL). In recent years it became clear that pathogenic herpesviruses including EBV, KSHV, and MHV68 express numerous long non-coding RNAs (lncRNAs) many of which are in antisense orientation to protein coding transcripts. The function and structure of these RNAs is largely unknown. In addition, these viruses express microRNAs (miRNAs). While characterizing the KSHV miRNA targetomes using a modified Crosslinking and Sequencing of Hybrids (qCLASH) protocol, we identified several hundred host cellular lncRNAs as putative miRNA targets. These data strongly suggest that both KSHV encoded proteins and miRNAs contribute to dysregulation of host lncRNAs. Importantly, 34 lncRNAs that are perturbed following KSHV infection, including MALAT1, HOTTIP, ANRIL, Meg3, UCA1 and GAS-5 are reported to be associated with human cancers. We also linked both mRNA and lncRNA targets to cancer hallmark phenotypes such as proliferation, migration, angiogenesis, and glucose metabolism, and started to identify signaling pathways that are perturbed by KSHV miRNAs in human endothelial cells as a model for KS. We also identified aberrant splicing as an additional cancer hallmark phenotype. Here we propose to extend our studies by integrating multi-omics data sets from qCLASH, RNAseq and miRNAseq data to comprehensively analyze miRNA-regulated gene regulatory networks in KSHV infected endothelial cells. Moreover, we propose to validate our findings in a significant number of human tumor samples and by generating the first KS tumor miRNA targetome by qCLASH. These studies will be performed in a comparative fashion with Projects 2 and 3 and furthermore will be supported by Cores B, C, and D. In addition, we are functionally studying the role of the antisense LANA transcript (ALT) for which we have identified 81 putative binding proteins using a highly innovative RNA-pulldown assay. Moreover, we will study the role of a newly identified class of viral circular RNAs that has been discovered by this program. In summary, the goal of this project is to delineate the role of viral lncRNAs and host cellular lncRNAs that are perturbed by viral infection in AIDS malignancies.