Fig. 1.

Schematic representation of the retargeted His-tagged MV-GFP-H_AA strain. The Y481A and R533A mutations in the hemagglutinin (H) gene ablate CD46 and SLAM interaction respectively (H_AA). The target receptor-binding ligand is displayed on the C-terminus of the ablated H_AA protein. The ligand is flanked by SfiI (encoding AAQPA amino acid sequence) and NotI (encoding AAA amino acid sequence) restriction sites. A six histidine peptide (H6) is attached to the C-terminus displayed ligand via a short linker sequence (AAARGS). H6 facilitates the “pseudoreceptor” interaction which allows propagation of the retargeted strain using Vero cells (Vero-aHis) generated to stably express a membrane-anchored single-chain antibody that binds to the H6 tag allowing virus entry and propagation. The FLAG-tag inserted after the start codon at the N-terminus of H_AA is used to facilitate detection by Western immunoblotting. Fxa is a factor Xa protease cleavage sequence (IEGR) that links the H_AA glycoprotein with the target receptor ligand peptide. It may be used to determine the recombinant nature of the chimeric H_AA (37). Thus, factor Xa protease cleavage allows removal of the peptide ligand resulting in an H_AA protein with similar molecular weight as that of unmodified virus. PacI/SpeI restriction enzymes are used to subclone the chimeric H_AA into the full-length infectious clone of the MV-NSe strain, deriving from MV-Edm. The virus also contains the gene encoding green fluorescent protein (eGFP) in position 1. (N, nucleoprotein gene; P, phosphoprotein gene; M, matrix protein gene; F, fusion protein gene; L, large protein gene; *, stop codons; standard one-letter abbreviations are used to denote amino acid residues). (modified from Nakamura T et al, Nature Biotechnology 23(2):209-14, 2005).