Receptor Trafficking in Entry of Murine Leukemia Viruses

描述(申请人提供)：在理解病毒进入方面的进展主要是由依赖于pH的病毒主导的，主要是因为通过降低pH可以同步进入，并且存在定量检测来测量这种集体融合事件。对逆转录病毒等pH不依赖的病毒的关注要少得多。该领域最重要的悬而未决的问题之一是，逆转录病毒在哪里穿透细胞膜，以及是否需要除受体相互作用以外的细胞信号来触发进入。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转运的重要基因的定制siRNA文库。我们使用包膜伪分型系统比较了Friend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。与肌动蛋白调节的rac1-PAK1-LIMK1途径相对应的基因被确定为感染Fr-MLV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个基因在内吞作用中起重要作用。由于伪型之间的唯一差异是所使用的包膜蛋白的来源，因此所看到的差异很可能是由于进入途径的不同。在这项建议中，我们将测试肌动蛋白和内吞作用对小鼠白血病病毒进入的作用。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。我们开发了一种新的病毒进入试验，可以实时测量病毒进入的动力学。该化验为入门测量提供了最高可能的细节。这个测试使我们能够定义进入的每个基因的角色，并允许我们区分在运输或内吞作用中的角色。这项研究将为包括艾滋病毒和包膜病毒在内的逆转录病毒的进入过程提供新的见解。对进入途径的了解反过来将有助于药物的开发，这些药物将阻断感染的第一步，并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.