Genetics of SV40 Entry and Minichromosome Transport

描述（由申请人提供）：猿猴病毒40 （SV40）通过结合质膜MHC I类分子感染细胞，然后通过由小泡介导的独特机制进入细胞，而不是由网格蛋白包裹的凹点。不寻常的SV40进入途径的一个显著特征是它将病毒转运到内质网（ER），而不是内体。重要的是，内质网是SV40内部衣壳蛋白VP2和VP3与病毒粒子分离的位点。VP2/3随后进入细胞质，而病毒粒子和主要衣壳蛋白VP1则留在内质网中。本项目的目的是研究SV40在细胞识别、进入和细胞内运输过程中的分子特征，并阐明SV40基因组（小染色体）的释放及其转运到细胞核的过程。具体目标如下。首先，确定SV40衣壳蛋白在SV40结合和细胞内转运中的作用，并确定这些蛋白质的分子特征，这些蛋白质是这种病毒不寻常进入和转运的基础。第二，识别。野生型SV40病毒粒子释放其小染色体的细胞区室，以及小染色体进入细胞核的途径。第三，表征VP2和VP3在脱膜小染色体和将其运出内质网过程中的功能。这里的一个特殊目的是确定多花型VP2是否在这些事件中起作用。第四，确定细胞骨架是否在将小染色体运送到细胞核中起作用。如果是这样，那么将进行实验来鉴定相关的运动蛋白，并评估VP2/3在细胞骨架介导的运输过程中的可能作用。

DESCRIPTION (provided by applicant): Simian virus 40 (SV40) infects cells by binding to plasma membrane MHC class I molecules and then entering via a unique mechanism mediated by caveolae, rather than clathrin-coated pits. A notable feature of the unusual SV40 entry pathway is that it transports the virus to the endoplasmic reticulum (ER), rather than to endosomes. Importantly, the ER is the site in which the SV40 internal capsid proteins VP2 and VP3 separate from virions. VP2/3 then enter the cytosol, whereas virions and the major capsid protein VP1 remain in the ER. The goal of this project is to investigate the molecular features of SV40 that underlie cell recognition, entry and intracellular trafficking, and to clarify the process of release of the SV40 genome (the minichromosome) and its transport to the nucleus. Specific objectives are as follows. First, to determine the roles of the SV40 capsid proteins in the binding and intracellular trafficking of SV40, and to identify the molecular features of those proteins that underlie the unusual entry and trafficking of this virus. Second, to identify the.cellular compartment.into which wild type SV40 virions release their minichromosomes and the route of the minichromosomes to the nucleus. Third, to characterize the function of VP2 and VP3 in the processes of uncoating the minichromosome and transporting it out of the ER. A particular aim here is to determine whether myristylated VP2 has a role in these events. Fourth, to determine whether the cellular cytoskeleton has a role in transporting the minichromosome to the nucleus. If it does, then experiments will be carried out to identify the relevant motor proteins and to evaluate the possible role of VP2/3 in the cytoskeleton-mediated transport process. Experimental procedures will make use of confocal microscopy, in which virions, virus proteins, and cellular organelles will be visualized by immunofluorescent staining. In some experiments, fluorescent in situ hybrization (FISH) will be combined with immunocytochemistry, to visualize minichromosomes with respect to cellular organelles, the cytoskeleton, and virus proteins. Experimental samples will include wild type virus, constructed virus mutants, and in vitro assembled virus particles that contain specific combinations of virus proteins. Recent studies report evidence that SV40 infection may lead to neoplastic disease in humans, including mesotheliornas, osteosarcomas, childhood brain tumors, and non-Hodgkin's Iymphoma.