Genetics of SV40 Entry and Minichromosome Transport

描述(申请人提供)：猴病毒40(SV40)通过与质膜MHC I类分子结合，然后通过一种独特的机制进入细胞，从而感染细胞，这种机制是由小凹而不是笼罩着网状蛋白的凹坑介导的。不寻常的SV40进入途径的一个显著特征是它将病毒运输到内质网(ER)，而不是内小体。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置。然后VP2/3进入胞浆，而病毒粒子和主要衣壳蛋白VP1留在内质网。这个项目的目的是研究SV40的分子特征，它是细胞识别、进入和细胞内转运的基础，并阐明SV40基因组(微染色体)的释放过程及其向细胞核的运输。具体目标如下。首先，确定SV40衣壳蛋白在SV40结合和细胞内运输中的作用，并确定这些蛋白的分子特征，这些蛋白是这种病毒异常进入和运输的基础。第二，确定SV40野生型病毒释放其微染色体进入的细胞室以及微染色体到达细胞核的途径。第三，研究VP2和VP3在微染色体脱壳和转运出内质网的过程中的作用。这里的一个特别目的是确定肉豆蔻酸化的VP2是否在这些事件中发挥作用。第四，确定细胞骨架是否在将微染色体运输到细胞核中起作用。如果是，那么将进行实验，以确定相关的马达蛋白，并评估VP2/3在细胞骨架介导的运输过程中可能发挥的作用。 实验程序将利用共聚焦显微镜，其中病毒粒子、病毒蛋白和细胞器将通过免疫荧光染色显示出来。在一些实验中，荧光原位杂交(FISH)将与免疫细胞化学相结合，以显示细胞细胞器、细胞骨架和病毒蛋白的微小染色体。实验样本将包括野生型病毒、构建的病毒突变体和包含特定病毒蛋白组合的体外组装的病毒颗粒。 最近的研究表明，SV40感染可能会导致人类的肿瘤性疾病，包括间皮瘤、骨肉瘤、儿童脑瘤和非霍奇金淋巴瘤。

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.