Regulation of Herpesvirus Infection by Viral miRNAs

描述（由申请方提供）：γ疱疹病毒，包括人gHV爱泼斯坦巴尔病毒和卡波西肉瘤相关疱疹病毒，感染免疫系统细胞并建立终身慢性感染，可能以良性潜伏状态存在。免疫功能下降（来自先天性、获得性或医源性免疫缺陷）是gHV相关疾病（包括肿瘤和慢性炎性疾病）的风险因素。我们对这些人类病原体的理解可以从感染和疾病的动物模型中取得重要进展。我们假设这些病毒调节RNA在这些相关的gHV中具有相似的功能，就像这些病毒中的不同病毒基因靶向共同的分子途径（如抗原呈递、细胞周期和细胞凋亡）一样。我们将1）在体外确定gHV 68 miRNAs的活性和需要，2）在体内测试病毒miRNAs对gHV 68感染和发病机制的遗传需要，和3）调查和鉴定gHV miRNAs的保守靶标。为了实现这一目标，我们将完成几种突变病毒的产生和验证，缺乏病毒miRNA或缺乏病毒靶序列，我们将利用酶标记的病毒来鉴定和纯化感染的细胞，我们将确定归因于病毒miRNA的整合蛋白质变化，最重要的是，我们将在整个动物感染的背景下进行这些研究。这些研究将使我们能够明确地解决病毒编码的miRNA对体内发病机制的遗传贡献，这是gHV发病机制的gHV 68模型唯一解决的问题。这些研究极有可能为gHV miRNA提供新的见解（例如，适合疫苗的病毒改变，治疗恢复的靶向途径的鉴定，或可能在其他疾病状态中有用的小RNA调节途径的鉴定）。

DESCRIPTION (provided by applicant): Gammaherpesviruses, including the human gHV Epstein Barr virus and Kaposi's sarcoma associated herpesvirus, infect cells of the immune system and establish lifelong, chronic infection that can exist in a benign, latent state. Decreased immune function (from congenital, acquired or iatrogenic immunedeficiencies) is a risk factor for gHV-associated diseases including tumors and chronic inflammatory disease. Important advances in our understanding of these human pathogens can be made from animal models of infection and disease. We hypothesize that these viral regulatory RNAs serve similar functions in these related gHV, much as divergent viral genes in these viruses target common molecular pathways (such as antigen presentation, cell cycle and apoptosis). We will 1) Define the activity of and requirement for the gHV68 miRNAs in vitro, 2) Test the genetic requirement of viral miRNAs to gHV68 infection and pathogenesis in vivo, and 3) Investigate and identify conserved targets of gHV miRNAs. To accomplish this, we will complete the generation and validation of several mutant viruses, lacking viral miRNAs or lacking a viral target sequence, we will make use of an enzymatically marked virus for identification and purification of infected cells, we will determine the integrated protein changes attributable to the viral miRNAs, and most importantly, we will conduct these studies in the context of whole animal infection. These studies will allow us to definitively address the genetic contribution of virally-encoded miRNAs to in vivo pathogenesis, a question uniquely addressed by the gHV68 model of gHV pathogenesis. These studies are highly likely to provide new insights into gHV miRNAs (e.g. virus alterations suitable for vaccines, identification of targeted pathways for therapeutic restoration, or identification of pathways for small RNA regulation that could be useful in other disease states).