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 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.
Thomsen
S
S
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Deutschland. thomsen@hpi.uni-hamburg.de
Vogt
B
B
von Laer
D
D
Heberlein
C
C
Rein
A
A
Ostertag
W
W
Stocking
C
C
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000502:physiology* / Q000821:virology; D009052:Leukemia Virus, Murine / Q000254:growth & development; D051379:Mice; D051381:Rats; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics*; D014161:Transduction, Genetic; D014779:Virus Replication
D004268:DNA-Binding Proteins; D011991:Receptors, Virus; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; C494185:Pou1f1 protein, mouse; C494186:Pou1f1 protein, rat; C067981:leukemia virus receptor, gibbon ape