LACK OF FUNCTIONAL PIT-1 AND PIT-2 EXPRESSION ON HEMATOPOIETIC STEM-CELL LINES

Hematopoietic stem cells (HSC) are an important target for retroviral gene transfer. However, transduction efficiency in these HSC is extrem ely low compared to fibroblasts or more mature hematopoietic cells. Th is infection block was analyzed in the HSC line FDC-Pmix. The infectio n frequency with the amphotropic murine leukemia virus (MLV-A) is more than 100-fold lower in FDC-Pmix cells as compared to fibroblasts. Pse udotyping with the env of the 10A1 strain (MLV-10A1), which uses both the amphotropic receptor (Pit-2) and the receptor for gibbon ape leuke mia virus (Pit-l), did not improve the infection efficiency. Vectors p seudotyped with VSV G protein were found to overcome the infection blo ck in FDC-Pmix, confirming that the block is at the level of virus bin ding and possibly penetration. Accordingly, we could not detect virus binding of MLV-A or MLV-10A1 to FDC-Pmix cell lines. Northern blot ana lysis was performed to detect whether the defect is at the level of tr anscription. Surprisingly, similar levels of Pit-2 receptor transcript s were detected in all cell types. The overexpression of rat Pit-2 DNA in CHO but not in FDC-Pmix cells improved amphotropic infection frequ ency after introducing rat Pit-2 DNA into the cells. Taken together th ese results show that the inefficient infection of FDC-Pmix is due to a lack of functional receptors. Either the receptor protein is incorre ctly processed in these cells or a cofactor is missing in FDC-Pmix cel ls that is necessary for efficient binding and/or penetration.