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.

项目概要 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，原发性 渗出性淋巴瘤 (PEL) 存在于所有肿瘤细胞中，并且在 > 95% 的感染细胞中紧密潜伏。 是一个组织复杂的程序，高度限制少数基因（即潜伏基因）的基因表达。 PEL 细胞系需要这些潜伏基因组成型表达，而裂解基因则被沉默 因此，潜伏基因的表达和潜伏程序的维持对于 PEL 的生存至关重要。 然而，大多数病毒基因对 PEL 衍生细胞系的存活和增殖的贡献。 我们的长期目标是加速我们对这种艾滋病定义的生物学的理解。 因此，我们通过综合分析 PEL 肿瘤细胞存活所需的病毒因素来研究淋巴瘤。 进行了 RNA 靶向 CRISPR/CasRx 平铺筛选，以确定生存所需的 KSHV 转录本 我们的结果表明，裂解复制的左侧起点（OriLytL）编码先前的 未注释的 RNA 对 PEL 细胞系的生存至关重要，OriLytL 是两个至关重要的起始位点之一。 在从潜伏期重新激活期间进行病毒 DNA 复制，OriLytL 产生两个长片段。 裂解病毒DNA复制所需的非编码RNA然而，我们的数据表明潜伏期特异性。 OriLytL (OriLytLlat) RNA 与裂解性 OriLytL RNA 不同，其功能也不同。 OriLytLlat RNA 的作用是维持潜伏期程序，因为潜伏期对于肿瘤的存活至关重要。 在细胞中，OriLytLlat RNA 是 KSHV 相关癌症的关键致癌驱动因素。 在目标 1 中，表征这种潜伏期特异性 OriLytLlat RNA 并确定其在潜伏期维持中的作用。 我们将使用长读长 RNA 来表征潜伏病毒转录组，包括全长 OriLytLlat RNA 在目标 2 中，我们将定义 OriLytLlat RNA 在招募染色质结合因子中的作用。 以及病毒基因组中由此产生的表观遗传变化，这些变化有助于建立和维持潜伏期 总之，这项工作将为病毒潜伏期如何受到潜伏 RNA 的影响提供一个新模型。 重要的是，我们的研究还将揭示其他 KSHV 相关的生物学。 恶性肿瘤，包括卡波西肉瘤。