Receptor Trafficking in Entry of Murine Leukemia Viruses

描述（由申请方提供）：在理解病毒进入方面的进展主要是由pH依赖性病毒主导的，主要是因为进入可以通过降低pH值同步进行，并且存在定量测定来测量这种进入融合事件。对非pH依赖性病毒如逆转录病毒的关注要少得多。这一领域最重要的未解决的问题之一是逆转录病毒穿透细胞膜的位置，以及是否需要细胞信号而不是受体相互作用来触发进入。为了鉴定对鼠白血病病毒进入重要的因素，我们设计并筛选了靶向对内吞作用和受体运输重要的基因的定制siRNA文库。我们比较感染的朋友鼠白血病病毒（Fr-MLV）水泡性口炎病毒，委内瑞拉马脑炎病毒和埃博拉病毒使用包膜假型系统。对应于肌动蛋白调节的Rac 1-PAK 1-LIMK 1途径的基因被鉴定为Fr-MLV感染的关键，而不是其他病毒。此外，发动蛋白，EEA 1和Eps 15 R被确定为重要的。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差异是所使用的包膜蛋白的来源，因此所观察到的差异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对小鼠白血病病毒进入的作用。肌动蛋白的作用可能是将受体运输通过细胞表面或进行内吞作用。我们开发了一种新的病毒进入试验，可真实的实时测量病毒进入动力学。该检测试剂盒可提供尽可能详细的进样测量结果。该测定使我们能够定义每个基因的进入作用，并使我们能够区分在运输或内吞作用中的作用。这项研究将提供新的洞察逆转录病毒，包括艾滋病毒和包膜病毒的进入过程中一般。对进入途径的了解将反过来有助于开发药物，这些药物将阻断感染的第一步并防止病毒的细胞间传播。

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.