To evaluate the ability of mutant 2D3 (derived from the rabies virus glycoprotein (RVG)-derived peptide, RDP) to deliver the anti-West Nile virus (WNV) single-chain variable fragment (scFv) into human neuroblastoma SH-SY5Y cells.

The cell-penetrating ability of 2D3 was tested using a green fluorescent protein (GFP) reporter system. The anti-West Nile virus single-chain variable fragment-2D3 fusion protein (single-chain variable fragment-2D3, scFv-2D3) was designed and expressed via genetic engineering. The antigen-binding activity and cell penetration into the target cells were assessed using enzyme linked immunosorbent assay (ELISA) and flow cytometry.

The 2D3 mutant showed stronger neurotropism and could more effectively mediate the uptake of the GFP reporter plasmid into SH-SY5Y cells than the parental RDP. The scFv-RDP could bind to West Nile virus envelope protein domain Ⅲ (WNV ED Ⅲ) in a concentration-dependent manner and the binding activity was comparable to that of the parental scFv without the penetrating peptide. scFv was detected intracellularly in SH-SY5Y cells treated with scFv-2D3, suggesting that 2D3 could efficiently deliver scFv into neuronal cells.

2D3 can more effectively deliver reporter plasmids and the anti-West Nile virus scFv into human neuroblastoma cells. This finding is expected to facilitate subsequent in vivo functional evaluation of the scFv-2D3 fusion protein.