Project 1: KSHV reprograms replication and metabolic activities in hypoxia

项目#1 摘要 KSHV相关癌症发生在低氧微环境中。低氧对病毒和宿主DNA的影响 对KSHV感染背景下的复制知之甚少。我们之前曾研究过复制 KSHV在常氧细胞培养体系中的致病作用。我们现在建议对KSHV和被感染的宿主细胞进行深入的分析 低氧环境中的复制。像疱疹病毒家族的许多成员一样，KSHV经历了 潜伏的和裂解的复制周期。在细胞培养系统中，潜伏期通过同步 病毒基因组的复制与宿主细胞的复制是细胞周期相关的。因此，大多数 潜伏复制活动在每个细胞周期内利用寄主机制一次。这些潜伏感染的细胞通常是 通过化学诱导剂处理诱导到裂解复制，并需要表达一些 病毒编码的基因。然而，关于病毒基因组在低氧条件下的复制以及病毒 调节病毒基因组持续存在所需的代谢变化所需的抗原通过 在受感染的细胞中进行复制，允许繁殖到新的子细胞中。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方法 与常氧相比，在低氧条件下发生的情况。我们将研究细胞复制的变化 细胞周期不同阶段的蛋白质及其翻译后修饰。它们在缺氧时的水平会 进行分析，以确定复制机制的基本组件的变化。此外，我们 将查看它们的转录以确定这些变化是否是由于 或者这些变化是由于缺氧时HIF1α对转录的调节所致。新陈代谢的变化 与KSHV阴性细胞相比，KSHV感染细胞在低氧条件下诱导的酶也将被分析。 以确定这些变化是否被转录重新编程。我们将确定新陈代谢基因 缺氧条件下KSHV感染的调控失调及复制基因是否受HIF1α的调控 低氧中的转录调节因子。我们将确定在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检测 模仿相关的病理。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，以确定这些蛋白在KSHV复制中的作用 缺氧的微环境。 14.

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