Thomsen S, Vogt B, von Laer D, Heberlein C, Rein A, Ostertag W, Stocking C
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Deutschland.
Acta Haematol. 1998;99(3):148-55. doi: 10.1159/000040829.
Hematopoietic stem cells (HSC) are an important target for retroviral gene transfer. However, transduction efficiency in these HSC is extremely low compared to fibroblasts or more mature hematopoietic cells. This infection block was analyzed in the HSC line FDC-Pmix. The infection frequency with the amphotropic murine leukemia virus (MLV-A) is more than 100-fold lower in FDC-Pmix cells as compared to fibroblasts. Pseudotyping with the env of the 10A1 strain (MLV-10A1), which uses both the amphotropic receptor (Pit-2) and the receptor for gibbon ape leukemia virus (Pit-1), did not improve the infection efficiency. Vectors pseudotyped with VSV G protein were found to overcome the infection block in FDC-Pmix, confirming that the block is at the level of virus binding and possibly penetration. Accordingly, we could not detect virus binding of MLV-A or MLV-10A1 to FDC-Pmix cell lines. Northern blot analysis was performed to detect whether the defect is at the level of transcription. Surprisingly, similar levels of Pit-2 receptor transcripts were detected in all cell types. The overexpression of rat Pit-2 DNA in CHO but not in FDC-Pmix cells improved amphotropic infection frequency after introducing rat Pit-2 DNA into the cells. Taken together these results show that the inefficient infection of FDC-Pmix is due to a lack of functional receptors. Either the receptor protein is incorrectly processed in these cells or a cofactor is missing in FDC-Pmix cells that is necessary for efficient binding and/or penetration.
造血干细胞（HSC）是逆转录病毒基因转移的重要靶点。然而，与成纤维细胞或更成熟的造血细胞相比，这些造血干细胞的转导效率极低。在造血干细胞系FDC-Pmix中分析了这种感染阻断情况。与成纤维细胞相比，双嗜性鼠白血病病毒（MLV-A）在FDC-Pmix细胞中的感染频率低100多倍。用10A1株（MLV-10A1）的env进行假型化，该毒株同时使用双嗜性受体（Pit-2）和长臂猿白血病病毒受体（Pit-1），并未提高感染效率。发现用VSV G蛋白假型化的载体可克服FDC-Pmix中的感染阻断，证实该阻断发生在病毒结合水平，可能还包括穿透水平。因此，我们无法检测到MLV-A或MLV-10A1与FDC-Pmix细胞系的病毒结合。进行Northern印迹分析以检测缺陷是否发生在转录水平。令人惊讶的是，在所有细胞类型中均检测到相似水平的Pit-2受体转录本。将大鼠Pit-2 DNA导入细胞后，在CHO细胞而非FDC-Pmix细胞中过表达大鼠Pit-2 DNA可提高双嗜性感染频率。综合这些结果表明，FDC-Pmix感染效率低下是由于缺乏功能性受体。要么是这些细胞中受体蛋白加工错误，要么是FDC-Pmix细胞中缺少高效结合和/或穿透所必需的辅助因子。
