Role of a latent OriLyt RNA in KSHV latency in primary effusion lymphoma

潜伏 OriLyt RNA 在原发性渗出性淋巴瘤 KSHV 潜伏期中的作用

• 批准号: 10761865
• 负责人: Mark Manzano
• 金额: $ 21.46万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10761865
• 关键词: Acceleration; Acquired Immunodeficiency Syndrome; Address; B lymphoid malignancy; B-Cell Lymphomas; Binding; Biology; Cell Line; Cell Survival; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; DNA biosynthesis; Data; Epigenetic Process; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Goals; HIV/AIDS; Hand; Human Herpesvirus 8; Kaposi Sarcoma; Left; Length; Life Cycle Stages; Lymphoma; Lymphoma cell; Lytic; Lytic Phase; Maintenance; Malignant Neoplasms; Modeling; Mutation; Oncogenic; Patients; Proliferating; Proteins; Public Health; RNA; RNA replication; Replication Origin; Research; Role; Site; Transcript; Untranslated RNA; Viral; Viral Genes; Viral Genome; Virus; Virus Latency; Work; histone modification; knock-down; latent gene expression; lytic replication; neoplastic cell; primary effusion lymphoma; programs; reactivation from latency; recruit; transcriptome; transcriptome sequencing; tumor; viral DNA

Project Summary The Kaposi's sarcoma-associated herpesvirus (KSHV) causes the AIDS-defining B cell malignancy, primary effusion lymphoma (PEL). KSHV is found in all tumor cells and is tightly latent in >95% of infected cells. Latency is an intricately organized program that highly restricts gene expression to a handful of genes (i.e. latency genes). PEL cell lines require these latency genes to be constitutively expressed while the lytic genes are silenced for survival. Thus, both expression of the latency genes and maintenance of the latency program are critical for PEL cells. However, the contributions of most viral genes to the survival and proliferation of PEL-derived cell lines are unknown. Our long-term goal is to accelerate our understanding of the biology of this AIDS-defining lymphoma by comprehensively analyzing viral factors required for PEL tumor cell survival. We, therefore, performed an RNA-targeting CRISPR/CasRx tiled screen to identify KSHV transcripts required for the survival of PEL cells. Our results reveal that the left-hand origin of lytic replication (OriLytL) encodes a previously unannotated RNA critical for the survival of PEL cell lines. The OriLytL serves as one of two initiation sites crucial for viral DNA replication during reactivation from latency. In this lytic phase, the OriLytL produces two long noncoding RNAs required for lytic viral DNA replication. However, our data suggest that the latency-specific OriLytL (OriLytLlat) RNA is distinct and functions differently from the lytic OriLytL RNAs. Our central hypothesis is that the OriLytLlat RNA acts to maintain the latency program. Since latency is crucial for the survival of tumor cells, the OriLytLlat RNA is a critical oncogenic driver of KSHV-associated cancers. The over-all objective is to characterize this latency-specific OriLytLlat RNA and determine its role in latency maintenance in PEL. In Aim 1, we will characterize the latent viral transcriptome, including the full-length OriLytLlat RNA using long-read RNA sequencing. In Aim 2, we will define the role of the OriLytLlat RNA in the recruitment of chromatin binding factors and resulting epigenetic changes in the viral genome that contribute to establish and maintain the latency program. Together, this work will provide a new model on how viral latency is influenced by a latent RNA from the lytic origin of replication. Importantly, our studies will also shed light on the biology of other KSHV-associated malignancies, including Kaposi’s sarcoma.

项目摘要 卡波西肉瘤相关疱疹病毒(KSHV)导致艾滋病定义的B细胞恶性肿瘤，原发 渗出性淋巴瘤(PEL)。KSHV存在于所有肿瘤细胞中，并在95%的感染细胞中潜伏。潜伏期 是一个组织复杂的程序，高度限制基因表达到少数几个基因(即潜伏基因)。 PEL细胞株需要这些潜伏基因的结构性表达，而裂解基因则被沉默 生死存亡。因此，潜伏期基因的表达和潜伏期程序的维护对于PEL来说都是至关重要的 细胞。然而，大多数病毒基因对PEL来源的细胞系的生存和增殖的贡献 都是未知的。我们的长期目标是加速我们对这种定义艾滋病的生物学的理解 通过综合分析PEL肿瘤细胞生存所需的病毒因素，研究淋巴瘤。因此，我们， 进行了RNA靶向CRISPR/CasRx平铺筛查以确定KSHV生存所需的转录本 PEL细胞的数量。我们的结果表明，裂解复制的左侧起点(OriLytL)编码一个先前的 未注解的RNA对PEL细胞系的生存至关重要。OriLytL是两个关键的起始点之一 用于从延迟重新激活期间的病毒DNA复制。在这个裂解阶段，OriLytL产生两个长的 裂解病毒DNA复制所需的非编码RNA。然而，我们的数据表明，特定于延迟的 OriLytL(OriLytLlat)RNA与裂解的OriLytL RNA不同，其功能也不同。我们的中心假设 是OriLytLlat RNA起到维持潜伏期程序的作用。因为潜伏期对肿瘤的存活率至关重要 在细胞中，OriLytLlat RNA是KSHV相关癌症的关键致癌驱动因素。总体目标是 鉴定这种潜伏期特异性的OriLytLlat RNA，并确定它在PEL潜伏期维持中的作用。在目标1中， 我们将使用长读RNA来表征潜伏的病毒转录组，包括全长的OriLytLlat RNA 测序。在目标2中，我们将确定OriLytLlat RNA在染色质结合因子招募中的作用 以及由此导致的病毒基因组的表观遗传学变化，这些变化有助于建立和维持潜伏期 程序。总之，这项工作将提供一个新的模型，说明病毒潜伏时间是如何受到来自 复制的裂解起源。重要的是，我们的研究也将揭示其他与KSHV相关的生物学 恶性肿瘤，包括卡波西肉瘤。