Analysis of host factors on neurotropism of SSPE virus

我们目前获得的结果表明SSPE菌株可能存在不同于CD150 （SLAM）和CD46的第三受体。本项目的目的是进一步证实该受体的存在，并克隆该分子的cDNA。为了证实其存在，采用VSV假型系统进行病毒进入实验，并用CD150或CD46特异性单克隆抗体阻断病毒进入。发现SSPE菌株使用CD150而不使用CD46，并且除了这两种分子外，还有一种特定的细胞表面分子。该分子在人神经母细胞瘤细胞和胶质瘤细胞中表达。作为病毒进入的另一个估计，用细胞松弛素D处理感染细胞制备的SSPE伪病毒体形成合胞体的情况也得到了与VSV伪病毒系统相同的结果。在此基础上，利用Vero细胞cDNA文库中的ecotropic逆转录病毒载体，对第3个受体进行cDNA克隆，该受体在我们所检测的细胞中表达最多。此外，制备阻断SSPE菌株进入Vero细胞的小鼠单克隆抗体，试图捕获新分子。目前，我们还没有成功，可能是因为它的表达量太少。这些工作目前仍在进行中。作为一种不同的方法，一些更敏感和有效的克隆方法正在考虑中。

Our results obtained so far suggested a possibility of the 3rd receptor for SSPE strains different from CD150 (SLAM) and CD46. The purpose of this project is to further confirm existence of this novel receptor and to clone cDNA of this molecule.To confirm its existence, virus entry assay was performed using VSV pseudotype system together with entry blocking by the specific monoclonal antibody against CD150 or CD46. SSPE strains were found to use CD150 but not CD46, in addition, a certain cell surface molecule except for the both ones. This novel molecule was expressed human neuroblastoma cells and human glioma cells. As another estimation on virus entry, syncytium formation by SSPE psedovirions prepared by treatment of the infected cells with cytochalasin D was monitored and the same results as VSV pseudotype system were also obtained.Based on the above confirmation, cDNA cloning of the 3rd receptor was tried using ecotropic retroviral vector from cDNA library of Vero cells, which seemed to express the molecule most among our available cells examined. In addition to it, preparation of mouse monoclonal antibody blocking entry of SSPE strains into Vero cells was tried to catch the novel molecule. At present, we have not yet succeeded probably because of too little amount of its expression. These are now still in progress. As a different approach, some more sensitive and effective cloning methods are under consideration.