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 卡波济肉瘤相关疱疹病毒（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）。KSHV存在于所有肿瘤细胞中，并且在>95%的感染细胞中紧密潜伏。延迟 是一个错综复杂的组织程序，高度限制基因表达的少数基因（即潜伏基因）。 PEL细胞系需要这些潜伏基因组成型表达，而裂解基因沉默， 生存因此，潜伏期基因的表达和潜伏期程序的维持对于PEL至关重要 细胞然而，大多数病毒基因对PEL衍生的细胞系的存活和增殖的贡献， 是未知的。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， 通过全面分析PEL肿瘤细胞存活所需的病毒因素，对恶性淋巴瘤进行了研究。因此，我们 进行了RNA靶向CRISPR/CasRx平铺筛选，以鉴定存活所需的KSHV转录物， 的PEL细胞。我们的研究结果表明，左侧裂解复制起点（OriLytL）编码一个先前的 对于PEL细胞系的存活至关重要的未注释的RNA。OriLytL作为两个关键的起始位点之一， 在潜伏期重新激活期间进行病毒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， OriLytL（OriLytLlat）RNA与裂解性OriLytL RNA不同并且功能不同。我们的核心假设 OriLytLlat RNA的作用是维持潜伏期程序。由于潜伏期对肿瘤的存活至关重要， OriLytLlat RNA是KSHV相关癌症的关键致癌驱动因子。总体目标是 表征这种潜伏期特异性OriLytLlat RNA并确定其在PEL中的潜伏期维持中的作用。在目标1中， 我们将使用长读段RNA来表征潜伏病毒的转录组，包括全长OriLytLlat RNA。 测序在目标2中，我们将定义OriLytLlat RNA在染色质结合因子募集中的作用 以及病毒基因组中导致的表观遗传变化，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，说明病毒潜伏期如何受到来自 复制的裂解起点重要的是，我们的研究也将揭示其他KSHV相关的生物学。 恶性肿瘤，包括卡波西肉瘤。