Entry of amphotropic and 10A1 pseudotyped murine retroviruses is restricted in hematopoietic stem cell lines

Abstract

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.

FIG. 1

Transduction of fibroblasts and hematopoietic progenitor cell lines with an A-MLV vector. Fibroblasts and hematopoietic cell lines were transduced at different MOIs with a retroviral vector containing neo packaged by an amphotropic helper virus. Transduction efficiencies were calculated as the ratio of G418-resistant cell clones to the number of unselected clones. Solid circles, SC-1 fibroblasts; open squares, NIH 3T3 cells; open triangles, L929 cells; open circles, cell line mentioned in the figure.

FIG. 1

Transduction of fibroblasts and hematopoietic progenitor cell lines with an A-MLV vector. Fibroblasts and hematopoietic cell lines were transduced at different MOIs with a retroviral vector containing neo packaged by an amphotropic helper virus. Transduction efficiencies were calculated as the ratio of G418-resistant cell clones to the number of unselected clones. Solid circles, SC-1 fibroblasts; open squares, NIH 3T3 cells; open triangles, L929 cells; open circles, cell line mentioned in the figure.

FIG. 2

Transduction of fibroblasts and hematopoietic progenitor cell lines with an MLV vector, pseudotyped with the surface proteins of 10A1. Fibroblasts and hematopoietic cell lines were transduced at different MOIs with a retroviral vector containing neo packaged by a 10A1 helper virus. Transduction efficiencies were calculated as the ratio of G418-resistant cell clones to the number of unselected clones. Solid circles, SC-1 fibroblasts; open squares, NIH 3T3 cells, open circles, cell line mentioned in the figure.

FIG. 2

Transduction of fibroblasts and hematopoietic progenitor cell lines with an MLV vector, pseudotyped with the surface proteins of 10A1. Fibroblasts and hematopoietic cell lines were transduced at different MOIs with a retroviral vector containing neo packaged by a 10A1 helper virus. Transduction efficiencies were calculated as the ratio of G418-resistant cell clones to the number of unselected clones. Solid circles, SC-1 fibroblasts; open squares, NIH 3T3 cells, open circles, cell line mentioned in the figure.

FIG. 3

Binding of amphotropic and 10A1 pseudotyped MLV to fibroblasts and hematopoietic cell lines. Cells were incubated with virus supernatants. Total virions, as well as the viral glycoprotein gp70^SU, bound to the cell surface were then stained with an anti-gp70 antibody, followed by binding of a biotinylated secondary antibody and streptavidin-phycoerythrin. The cells were then analyzed on a flow cytometer.

FIG. 4

Transduction of fibroblasts and FDC-Pmix cells with an amphotropic and a VSV G protein-pseudotyped vector. Cells were transduced at different MOIs with a retroviral vector containing neo packaged in the amphotropic packaging cell line GP+Am12 (A) or as VSV G-protein pseudotypes in the packaging line 293gp2 (B). Transduction efficiencies were calculated as the ratio of G418-resistant cell clones to the number of unselected clones. Solid circles, SC-1 fibroblasts; open circles, cell line mentioned in the figure.