Cell Entry and Spread by Polyoma Virus

描述（由申请人提供）：本申请的长期目标是了解小鼠多瘤病毒进入细胞的机制。这种病毒的宿主范围很广。它在其自然宿主中复制并诱导多种细胞类型的肿瘤。具体目的是首先确定某些糖脂是否构成病毒的唯一一类细胞受体，或者某些糖蛋白是否也可以作为功能性受体。这将用糖脂生物合成缺乏但糖蛋白途径不受影响的小鼠来完成。将进行调查，以确定病毒在内质网中的分解步骤和病毒利用的细胞因子进行必要的分解步骤并进入细胞核。在这方面正在研究的因素是氧化还原酶ERp29，蛋白质二硫异构酶，德林伴侣和Ran GTPase。一个特定的目标是在体外产生并表征改变的颗粒，这些颗粒为膜插入和从内质网逃逸做好了准备。最后，在与哈佛大学庄晓伟博士实验室的合作下，将尝试跟踪活细胞中的单个病毒颗粒，直到它们进入并建立感染。我们将尝试用荧光探针制备同时标记在小染色体和外衣壳蛋白上的多瘤颗粒，研究这些成分的脱壳和分离。许多病毒进入细胞的过程尚不完全清楚。多瘤组包括几种对人类致病的病毒，包括JC、BK和可能的SV40。了解这些病毒进入细胞的途径可能为抗病毒治疗或预防提供机会。

DESCRIPTION (provided by applicant): The long-term objectives of this application are to understand the mechanisms of cell entry by the mouse polyoma virus. This virus has a broad host range. It replicates and induces tumors in a wide variety of cell types in its natural host. The Specific Aims are first to determine whether certain glycolipids constitute the only class of cell receptors for the virus or whether certain glycoproteins may also serve as functional receptors. This will be done using a mouse deficient in glycolipid biosynthesis but unaffected in glycoprotein pathways. Investigations will be carried out to identify the steps in virus disassembly in the endoplasmic reticulum and the cellular factors the virus utilizes to undergo the necessary steps of disassembly and to enter the nucleus. Factors under investigation in this regard are the oxidoreductase ERp29, protein disulfide isomerase, the derlin chaperones, and Ran GTPase. A specific goal is to generate in vitro and characterize the altered particle that is primed for membrane insertion and escape from the ER. Finally, in collaboration with Dr. Xiaowei Zhuang's lab at Harvard, attempts will be made to follow single virus particles in live cells as they enter and establish infection. We will attempt to produce polyoma particles labeled both on the minichromosome and the outer capsid protein with fluorescent probes to study decapsidation and separation of these components. The processes of cell entry by many viruses are not fully understood. The polyoma group includes several viruses that are pathogenic in humans, including JC, BK and possibly SV40. Understanding the pathways of cell entry by these viruses may suggest opportunities for anti-viral therapy or prevention.