title: Interaction of vesicular stomatitis virus-G pseudotyped retrovirus with CD34+ and CD34+ CD38- hematopoietic progenitor cells.
abstract: 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. Virus binding to CD34+ cells was saturable at 4 degrees C but nonsaturable at 37 degrees C, 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-stimulated CD34+ CD38- cells has occurred, virus fusion proceeds efficiently 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.
authors.affiliation: Department of Haematology, University of Cambridge, MRC Center, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Sinclair
authors.forename: Y P
authors.initials: YP
authors.lastname: Agrawal
authors.forename: E
authors.initials: E
authors.lastname: Elbar
authors.forename: R
authors.initials: R
authors.lastname: Agrawal
authors.forename: A D
authors.initials: AD
authors.lastname: Ho
authors.forename: F
authors.initials: F
authors.lastname: Levine
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D017304:Annexin A5 / Q000378:metabolism; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000276:immunology*; D006801:Humans; D008562:Membrane Glycoproteins; D009244:NAD+ Nucleosidase; D012190:Retroviridae / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus; D014777:Virus Diseases / Q000628:therapy
chemical_list: D017304:Annexin A5; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1