Figure 1: Display of pen on rpVIII mediates receptor-independent cell internalization.
From: Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells
(a) Upper: parental f88-4 phage vector contains two capsid genes encoding a wild-type (wt) protein VIII (pVIII, depicted in grey) and a recombinant protein VIII (rpVIII; depicted in green). The recombinant gene VIII contains a foreign DNA insert with a HindIII and a PstI cloning site (depicted in red). TetR, tetracycline resistance gene. Lower: representation of the assembled phage particle expressing only the wt major coat protein pVIII (grey); pIII, minor coat protein (orange); pVI protein (blue); pVII protein (red), and pIX protein (yellow). (b) Upper: annealed oligonucleotides encoding the pen peptide were cloned in frame with the recombinant gene VIII. Lower: iPhage particles displaying the pen peptide motif (RQIKIWFQNRRMKWKK) at the amino terminus of the rpVIII (green). (c) Upper: the mutant iPhage genome has nucleotide substitutions in the pen sequence replacing tryptophan (W) by alanine (A) residues (underlined in red). Lower: representation of the assembled mutant iPhage displaying mutant pen on rpVIII protein (purple). (d) Immunofluorescence of KS1767 cells shows internalized viral particles only in cells incubated with iPhage. The nuclear stain DAPI emits blue fluorescence, and internalized phage particles were detected with conjugated antibodies (red fluorescence). Scale bar, 100 μm. (e) Phage genomic DNA was detected only in KS1767 cells incubated with iPhage particles (Southern blot, upper panel). Total genomic DNA stained with ethidium bromide served as a loading control (lower panel). (f) iPhage particles are internalized and viable in the cytosol and undetected in the membrane fraction of KS1767 cells. Bars represent mean values for TU recovered from the cytosol±s.e.m., from triplicates. Phage internalization by various types of cultured cells (g) mouse and (h) human. RMA (Rauscher murine leukaemia virus antigen) lymphoma, LLC (Lewis lung carcinoma), B16F10 (Melanoma), HUVEC (human umbilical vein endothelial cell), leukaemia (K562), 293HEK (human embryonic kidney) and HeLa cell lines. Internalization assays were run in triplicate; bars represent mean values for phage TU recovered from the cytosol-enriched fraction±s.e.m. from triplicates.
