Project 1: KSHV reprograms replication and metabolic activities in hypoxia

项目 1：KSHV 在缺氧条件下重新编程复制和代谢活动

• 批准号: 10714173
• 负责人: ERLE S. ROBERTSON
• 金额: $ 46.85万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714173
• 关键词: Address; Animal Model; Antigens; Automobile Driving; B-Lymphocytes; Biological Assay; Cell Culture System; Cell Cycle; Cell Cycle Proteins; Cell Hypoxia; Cell Line; Cell Proliferation; Cell Reprogramming; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoprotection; DNA Viruses; DNA biosynthesis; Data; Development; Drug Targeting; Endothelial Cells; Environment; Enzyme Induction; Enzymes; Epigenetic Process; Etiology; Family; Generations; Genes; Genetic Transcription; Genome; Goals; HIF1A gene; Harvest; Herpesviridae; Herpesviridae Infections; Human; Human Cell Line; Human Herpesvirus 8; Hypoxia; Immunocompromised Host; In Vitro; Incubated; Individual; Kaposi Sarcoma; Knock-out; Lesion; Link; Lymphoma; Lymphoproliferative Disorders; Malignant Neoplasms; Mediating; Metabolic; Metabolism; Modification; Molecular; Multicentric Angiofollicular Lymphoid Hyperplasia; Nucleotides; Oncogenic Viruses; Oncornaviruses; Pathogenicity; Pathology; Pathway interactions; Phase; Pleural effusion disorder; Population; Post Translational Modification Analysis; Post-Translational Protein Processing; Predisposition; Production; Proteins; Proteomics; Regulation; Role; Stress; System; Tetanus Helper Peptide; Time; Transcriptional Regulation; Translations; Viral; Viral Antigens; Viral Genome; Virus; Virus Latency; Virus Replication; Western Blotting; daughter cell; infected B cell; insight; knock-down; lytic replication; member; normoxia; novel; primary effusion lymphoma; programs; small hairpin RNA; three dimensional cell culture; transcriptome; tumorigenesis; virus related cancer

Project #1 Abstract KSHV-associated cancers occur in hypoxic microenvironments. The effects of hypoxia on viral and host DNA replication in the context of KSHV infection are poorly understood. We have previously investigated the replication of KSHV in normoxia cell culture systems. We now propose an in-depth analysis of KSHV and infected host cell replication in the hypoxic environment. KSHV, like many members of the herpesvirus family undergoes both a latent and a lytic replication cycle. In cell culture systems, latency is maintained through synchronization of the viral genome replication with that of the host cell replication, which is cell cycle dependent. Therefore, most of the latent replication activities utilize the host machinery once per cell cycle. These latently infected cells are typically induced to lytic replication through treatment with chemical inducers and requires the expression of a number of viral encoded genes. However, little is known regarding replication of the virus genome in hypoxia, and the viral antigens needed to regulate the ongoing metabolic changes required for persistence of the viral genome through replication in infected cells that allow for propagation into new daughter cells. We will approach these questions through a number of aims geared towards understanding the changes in the cellular and viral replication proteins that occur in hypoxia compared to that seen in normoxia. We will examine the changes in cellular replication proteins at different phases of the cell cycle, and their post-translational modification. Their levels in hypoxia will be analyzed to determine the changes in the essential components of the replication machinery. In addition, we will look at their transcription to determine if these changes are due to changes in the overall transcription of the genes or if the changes are due to transcription modulation by HIF1α in hypoxia. The changes in metabolic enzymes that are induced in KSHV infected cells compared to KSHV negative cells in hypoxia will also be analyzed to determine if the changes are transcriptionally reprogrammed. We will determine the metabolic genes that are dysregulated on KSHV infection in hypoxia and if the replication genes are regulated by HIF1α, the master transcription regulator in hypoxia. We will determine the epigenetic changes that occur during hypoxia that will determine the expression of the replication genes. We will also perform these assays in 3D culture systems to mimic the associated pathologies. We will also generate shRNA knockdown or CRISPR knockout of selected replication genes, and Tet-regulated genes to determine the contribution of these proteins in KSHV replication in the hypoxic microenvironment. 14

项目#1 抽象的 KSHV 相关癌症发生在缺氧微环境中。缺氧对病毒和宿主DNA的影响 人们对 KSHV 感染背景下的复制知之甚少。我们之前已经研究过复制 KSHV 在常氧细胞培养系统中的作用。我们现在建议对 KSHV 和感染的宿主细胞进行深入分析 在缺氧环境下进行复制。 KSHV 与疱疹病毒家族的许多成员一样，经历了 潜伏和裂解复制周期。在细胞培养系统中，延迟是通过同步来维持的 病毒基因组复制与宿主细胞复制相同，这是细胞周期依赖性的。因此，大多数 潜在的复制活动在每个细胞周期利用宿主机器一次。这些潜伏感染的细胞通常是 通过化学诱导剂处理诱导裂解性复制，并且需要表达许多 病毒编码基因。然而，人们对缺氧条件下病毒基因组的复制以及病毒的复制知之甚少。 调节病毒基因组持续存在所需的持续代谢变化所需的抗原 在受感染的细胞中进行复制，从而繁殖成新的子细胞。我们将探讨这些问题 通过一系列旨在了解细胞和病毒复制蛋白变化的目标 与常氧条件下相比，缺氧条件下发生的情况。我们将检查细胞复制的变化 细胞周期不同阶段的蛋白质及其翻译后修饰。他们的缺氧水平会 进行分析以确定复制机器的基本组件的变化。此外，我们 将查看它们的转录以确定这些变化是否是由于整体转录的变化引起的 基因或变化是否是由于缺氧时 HIF1α 的转录调节所致。新陈代谢的变化 还将分析 KSHV 感染细胞与 KSHV 阴性细胞在缺氧条件下诱导产生的酶 以确定这些变化是否是转录重编程的。我们将确定代谢基因 缺氧条件下 KSHV 感染失调，如果复制基因受 HIF1α 调节，则主控基因 缺氧时的转录调节因子。我们将确定缺氧期间发生的表观遗传变化 确定复制基因的表达。我们还将在 3D 培养系统中进行这些测定 模仿相关的病理。我们还将生成选定的 shRNA 敲低或 CRISPR 敲除 复制基因和 Tet 调节基因，以确定这些蛋白质在 KSHV 复制中的贡献 缺氧微环境。 14