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

基本信息

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
项目类别：
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

项目摘要

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。我们之前对 KSHV 感染情况下的复制情况了解甚少。我们现在建议对 KSHV 和感染的宿主细胞进行深入分析。 KSHV 与疱疹病毒家族的许多成员一样，在缺氧环境中进行复制。在细胞培养系统中，潜伏期和裂解性复制周期是通过同步来维持的。病毒基因组的复制与宿主细胞的复制一样，因而大多是细胞周期依赖性的。潜伏的复制活动通常在每个细胞周期利用宿主机器一次。通过化学诱导剂处理诱导裂解性复制，并且需要表达许多然而，对于病毒基因组在缺氧条件下的复制以及病毒的编码基因知之甚少。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，从而繁殖到新的子细胞中，我们将解决这些问题。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发生的情况相比，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在缺氧条件下的水平。进行分析以确定复制机器的基本组件的变化。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。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，我们将确定这些变化的代谢基因。缺氧条件下 KSHV 感染失调，如果复制基因受 HIF1α 调节，则主控基因我们将确定缺氧期间发生的表观遗传变化。我们还将在 3D 培养系统中进行这些测定，以确定复制基因的表达。我们还将生成选定的 shRNA 敲除或 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