INTERACTION OF VESICULAR STOMATITIS VIRUS-G PSEUDOTYPED RETROVIRUS WITH CD34(-) HEMATOPOIETIC PROGENITOR CELLS() AND CD34(+)CD38()

Retroviral vectors have had limited success in mediating gene transfer to hematopoietic stem cells, particularly in primates, due in part to low or absent expression of the amphotropic receptor (RAM-1). We have been interested in determining whether retrovirus pseudotyped with ve sicular stomatitis virus G protein (VSV-G) would allow more efficient gene delivery to hematopoietic stem cells as the VSV-G receptors appea r to be ubiquitously present phospholipids. However, we previously fou nd that completion of retroviral vector reverse transcription does not occur in CD34(+)CD38(-) hematopoietic stem cells that were exposed to VSV-G pseudotyped retrovirus. To determine at which stage the block t o infection of CD34(+)CD38(-) cells occurs, we confirmed by FACS analy sis that VSV-G pseudotyped viral particles could bind to CD34(+)CD38(- ) cells occurs, we confirmed by FACS analysis that VSV-G pseudotyped v iral particles could bind to CD34(+)CD38(-) cells. Virus binding to CD 34(+) cells was saturable at 4 degrees D, up to a multiplicity of infe ction of 1080. This suggests that surface levels of phospholipid recep tors available for viral binding are limiting on CD34(+) cells. Cytoki ne stimulation increased virus binding to CD34(+) cells. However, no i ncrease in the level of surface phosphatidylserine (PS), a strong cand idate for the VSV-G receptor, was seen as detected by the PS-specific reagent, annexin V. This suggests that another molecule is serving as the VSV-G receptor on CD34(+) cells. Here, we show that once virus bin ding to cytokine-stimulate CD34(+)CD38(-) cells has occurred, virus fu sion proceeds efficently as determined by octadecyl rhodamine (R18) fu sion assays. Taken together with our previous observation that reverse transcription does not occur in CD34(+)CD38(-) cells, we suggest that there are intracellular mechanisms leading to blockage of complete re verse transcription of the retrovirus in CD34(+)CD38(-) cells. This ha s important implications for retrovirus-mediated gene transfer to quie scent stem cells.