(A) Construct design. The aa sequence of VPR and its mutant version generated is shown. VPR, a small HIV-1 accessory protein, is carried in viral particles bound to gag via residues in its central region that fold into 3 α-helices. The C-terminal domain has 6 arginine residues that potentiate nuclear localization, G₂M arrest, and apoptosis. S79 phosphorylation is important for cell cycle arrest. We truncated VPR at the 78 aa and mutated VPR^R77Q. W54 and Q65 interacts with DNA damage response (DDR) proteins via UNG2 and DCAF and were mutated to VPR^W54R and VPR^Q65R. The triple mutated and truncated VPR (VPR^MT) would lack pathogenicity (residues known to be associated with VPR toxicity are highlighted in red) but allow binding to gag. VPR^MT was fused to CXCR4 cDNA via the HIV-1 protease cleavage site (PCS) to generate VPR^MT-CXCR4. (B) K562 cells were transduced with LV^CXCR4 vector-like particles (VLP; empty vector particles lacking the vector genome) at increasing particle concentration and analyzed for CXCR4 expression using flow cytometry. Mean fluorescence intensity (MFI) of CXCR4 is listed against volume of VLP added. (C and D) A GFP-encoding LV was either packaged using standard packaging plasmids (LV, black) or packaged with VPR^MT-CXCR4 plasmid in addition in order to package the CXCR protein attached to the LV capsid (LV^CXCR4, red). CXCR4 expression on MPB CD34⁺ cells transduced with LV^CXCR4 vector compared with cells transduced with the control LV vector 24 hours following gene transfer is shown. n = 4; statistical analysis was performed by Mann-Whitney U test. (E) The time course of CXCR4 expression in LV^CXCR4 HSPC, normalized to that of control LV HSPC, is shown, with expression peaking at 24 hours that returns to baseline by 72 hours. Symbols represent individual MPB donors; n = 3.