Functional display of TPO and SCF on HIV-derived vectors. (A) BAF3-Mpl cells expressing the TPO receptor were incubated with LVs presenting either of 2 different truncated forms of TPO at their surface. The TPO constructs were obtained by fusing the first 163 or 171 amino acids of TPO to the N-terminus of the amphotropic MLV glycoprotein (G/163SU and G/171SU) or by fusing the first 163 amino acids of TPO to the N-terminus of the influenza HA glycoprotein (G/TPOHA). The SCF construct was generated by fusing SCF to the N-terminus of the amphotropic MLV glycoprotein (SCFSUx). Incubations were also performed with vectors codisplaying TPO and SCF. After a 72-hour incubation with decreasing doses of vector (MOI ranging from 20 to 1), cell survival was determined and compared to that observed following incubation with VSV-G–displaying vectors alone or with rTPO (100 ng/mL to 5 ng/mL). G/TPOHA vectors were concentrated (G/TPOHA-conc) by ultracentrifugation and incubations were performed under the same conditions as for nonconcentrated TPO-displaying vectors. (B) BAF3-cKit cells expressing the SCF receptor were incubated with LVs presenting SCF at their surface by fusion to the amphotropic MLV glycoprotein (G/SCFSUx) or copresenting TPO and SCF at their surface. After a 72-hour incubation with decreasing doses of virus (MOI ranging from 20 to 1), cell survival was determined and compared to that observed following incubation with VSV-G–displaying vectors alone or with rSCF (100 ng/mL to 5 ng/mL). G/SCFSUx vectors were concentrated (G/SCFSUx-conc) by ultracentrifugation and incubations were performed under the same conditions as for nonconcentrated SCF-displaying vectors. (C) Immunoblots of LV particles displaying TPOHA or SCFSUx or both chimeric glycoproteins at their surface. Virions were purified over a sucrose cushion by ultracentrifugation. The upper part of the membrane was stained with antibodies against the influenza HA glycoprotein, the middle section with antibodies against MLV-SU to detect the TPOHA and SCFSUx chimeric envelopes, respectively. The lower part of the membrane was stained with antibodies against HIV-1 capsid to assess equivalent loading of purified vectors. The positions of the chimeric precursor protein (TPOHA), its processed isoform (TPOHA1), the SCFSUx protein, and the HIV capsid are indicated. (D) CD34⁺ CB cells were incubated for 72 hours with the indicated TPO-, SCF-, or TPO/SCF-displaying vectors at MOIs of 20 or 4. Survival of the cells was determined by PI staining. As control, CD34⁺ cells were incubated with vectors displaying VSV-G in the absence (–) or in the presence of cytokines (rTPO = 10 ng/mL; rSCF = 50 ng/mL). Data are shown as means ± SD, n = 4.