Receptor Trafficking in Entry of Murine Leukemia Viruses

描述（由申请人提供）：了解病毒进入的进展主要是pH依赖性病毒，主要是因为进入可以通过降低pH来同步，并且存在定量分析来测量这种大规模融合事件。对诸如逆转录病毒等不依赖ph值的病毒的关注要少得多。这一领域最重要的尚未解决的问题之一是逆转录病毒在哪里穿透细胞膜，以及是否需要细胞信号（而不是受体相互作用）来触发进入。为了确定小鼠白血病病毒进入的重要因素，我们设计并筛选了一个针对内吞作用和受体运输重要基因的定制siRNA文库。我们使用包膜假分型系统比较Friend小鼠白血病病毒（Fr-MLV）与水泡性口炎病毒、委内瑞拉马脑炎病毒和埃博拉病毒的感染情况。与肌动蛋白调控途径Rac1-PAK1-LIMK1相对应的基因被鉴定为Fr-MLV感染的关键基因，而不是其他病毒。此外，Dynamin、EEA1和Eps15R也被认为是重要的。后3个基因在胞吞作用中起重要作用。由于假型之间的唯一区别是所使用的包膜蛋白的来源，因此所见的差异很可能是由于进入途径的差异。在这个提议中，我们将测试肌动蛋白和内吞作用在小鼠白血病病毒进入中的作用。肌动蛋白的作用可能是通过细胞表面运输受体或内吞作用本身。我们已经开发了一种新的病毒进入试验，可以实时测量病毒进入动力学。该分析为入口测量提供了尽可能高的细节。这种测定方法使我们能够确定每个基因进入的作用，并使我们能够区分在运输或内吞作用中的作用。这项研究将为包括HIV和包膜病毒在内的逆转录病毒的进入过程提供新的见解。了解进入途径将有助于开发药物，阻止感染的第一步，防止病毒在细胞间传播。

DESCRIPTION (provided by applicant): Progress in understanding virus entry has been dominated by pH-dependent viruses, principally because entry can be synchronized by lowering pH and quantitative assays exist to measure this en masse fusion event. Much less attention has been paid to pH-independent viruses such as retroviruses. One of the most important unresolved issues in this field is where retroviruses penetrate the cell membrane and if cellular cues, other than receptor interaction, are required to trigger entry. To identify factors important for Murine leukemia virus entry we designed and screened a custom siRNA library targeting genes important for endocytosis and receptor trafficking. We compared infection of Friend murine leukemia virus (Fr-MLV) to Vesicular stomatitis virus, Venezuelan equine encephalitis virus and Ebola using an envelope pseudotyping system. Genes corresponding to the Rac1-PAK1-LIMK1 pathway of actin regulation were identified as key for infection by Fr-MLV but not the other viruses. Also, Dynamin, EEA1 and Eps15R were identified as important. The latter 3 genes play important roles in endocytosis. Since the only difference between the pseudotypes is the source of envelope protein used, it is likely that the differences seen were due to differences in entry pathway. In this proposal we will test the role of the actin and endocytosis for Murine leukemia virus entry. The role of actin may be for trafficking of receptor across the cell surface or endocytosis itself. We have developed a new virus entry assay that measures virus entry kinetics in real time. The assay provides the highest possible detail for entry measurements. This assay allows us to define the role of each gene for entry and permits us to distinguish roles in trafficking or endocytosis. This study will provide new insight into the entry process of retroviruses including HIV and enveloped viruses in general. Knowledge of the entry pathway will in turn aid in development of drugs that will block this first step in infection and prevent cell to cell spread of virus.