Cell Type Specific Outcomes of Gammaherpesvirus Infection.

描述(由申请人提供)：潜伏的伽马疱疹病毒(？HV)感染存在于世界上90%的人口中，并与慢性炎症和许多恶性肿瘤有关。关于初次感染的细胞结局的知识有很大的差距，这限制了预防潜伏感染的治疗方法的发展。我们假设，原发HV感染的结局取决于感染的细胞类型。本文概述的实验将以小鼠伽玛疱疹病毒68(？HV68)为模型，增强对HV感染早期事件的认识。我们将重点研究B细胞和内皮细胞，因为它们都在慢性HV感染中起作用，并且是许多HV相关肿瘤的转化细胞类型。我们的初步数据表明，yHV68感染在B细胞和内皮细胞中的结局非常不同，而且这两种感染都不符合目前裂解复制与潜伏感染的工作模式。本项目的目的1是研究yHV68在B细胞中感染的早期结局。通过对体外和体内感染的分析，我们将利用标记病毒来确定yHV68基因组定位到受感染B细胞的细胞核的效率。我们还将通过荧光素酶报告和PCR分析来确定yHV68基因组在受感染的B细胞中转录的强健程度。本项目的目标2是确定自噬对体外EC感染结局的贡献。我们将确定yHV68感染的EC幸存者是否正在接受自噬，以及抑制自噬对感染结局有什么影响。我们还将测试候选自噬相关病毒基因在EC感染结局中的作用。本项目的目的3是研究yHV68感染在体内的EC结局。我们将使用免疫组织化学、原位杂交和RT-PCR来检测和鉴定感染小鼠组织中的感染内皮细胞。相关性：慢性yHV感染存在于90%的人群中，并与许多不同癌症的发生有关。目前，我们对yHV感染的早期结果知之甚少，无法设计药物来降低慢性疾病相关感染的患病率。这些实验很重要，因为它们将确定相关特定细胞类型中yHV感染的早期结果。

DESCRIPTION (provided by applicant): Latent gammaherpesvirus (?HV) infection exists in >90% of the world's population, and is associated with chronic inflammation and numerous malignancies. There is a significant gap in knowledge regarding cellular outcomes of primary infection, and this limits the development of therapeutics to prevent latent infection. We hypothesize that outcome of primary ?HV infection is dependent on the cell type infected. The experiments outlined here will enhance knowledge of the early events in ?HV infection, using murine gammaherpesvirus 68 (?HV68) as a model. We will focus our investigation on B cells and endothelial cells (ECs) because they both have roles in chronic ?HV infection and are the transformed cell types of many ?HV-associated tumors. Our preliminary data indicate that outcome of yHV68 infection is very different in B cell and ECs, and that neither infection adheres to the current working model of lytic replication versus latent infection. Aim 1 of this project is to investigate the early outcome of yHV68 infection in B cells. Analyzing both in vitro and in vivo infection, we will make use of labeled virus to determine how efficiently yHV68 genome localizes to the nucleus of infected B cells. We also will determine how robustly yHV68 genome is transcribed in infected B cells via a luciferase reporter and PCR analysis. Aim 2 of this project is to determine the contribution of autophagy to in vitro EC infection outcome. We will determine if EC survivors of yHV68 infection are undergoing autophagy, and what effect inhibiting autophagy has on infection outcome. We also will test candidate autophagy-associated viral genes for their role in EC infection outcome. Aim 3 of this project is to characterize EC outcome of yHV68 infection in vivo. We will detect and characterize infected ECs in tissues from infected mice using immunohistochemistry, in situ hybridization, and RT-PCR. Relevance: Chronic yHV infection exists in >90% of the population and is associated with the development of many different cancers. Currently we do not know enough about the early outcomes of yHV infection to design drugs for reducing the prevalence of chronic, disease-associated infection. These experiments are important because they will determine early outcomes of yHV infection in relevant specific cell types.