Interaction of vesicular stomatitis virus-G pseudotyped retrovirus with CD34(+) and CD34(+)CD38(-) hematopoietic progenitor cells

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 vesicular stomatitis virus G protein (VSV-G) would allow more efficient gene delivery to hematopoietic stem cells as the VSV-G receptors appear to be ubiquitously present phospholipids. However, we previously found 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 to infection of CD34(+)CD38(-) cells occurs, we confirmed by FACS analysis that VSV-G pseudotyped viral particles could bind to CD34(+)CD38(-) cells occurs, we confirmed by FACS analysis that VSV-G pseudotyped viral particles could bind to CD34(+)CD38(-) cells. Virus binding to CD34(+) cells was saturable at 4 degrees D, up to a multiplicity of infection of 1080. This suggests that surface levels of phospholipid receptors available for viral binding are limiting on CD34(+) cells. Cytokine stimulation increased virus binding to CD34(+) cells. However, no increase in the level of surface phosphatidylserine (PS), a strong candidate 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 binding to cytokine-stimulate CD34(+)CD38(-) cells has occurred, virus fusion proceeds efficently as determined by octadecyl rhodamine (R18) fusion 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 reverse transcription of the retrovirus in CD34(+)CD38(-) cells. This has important implications for retrovirus-mediated gene transfer to quiescent stem cells.