Schematic diagram of all Ad vectors constructed and used in our study. (A) Cloning strategy to design protein IX-DAF and pIX-DAF_REO is shown. Nucleotide (top) and amino acid (bottom) sequences are depicted, corresponding to 5 C-terminal amino acids of protein IX fused in-frame (using NheI enzyme, recognition sequence of which adds 2 amino acids “ALA-SER” flanking DAF or DAF_REO sequence), followed by DAF or DAF_REO sequence and stop codon. Only first and last 10 amino acids of human DAF or DAF_REO are shown. Note that natural display of human DAF protein from cell surface allows N-terminus to be exposed; however, when DAF is displayed from pIX in its natural form, C-terminus is protruding from Ad5 capsid and therefore oppose natural orientation of DAF. To overcome this limitation we have designed Ad5 vector capsid-displaying retro form of DAF (amino acids 35-354), which mimic natural orientation of DAF with N-terminus protruding from Ad5 capsid. Note that N-terminal amino acids for DAF become C-terminal for DAF_REO and vice versa. Numbers represent amino acids of native human DAF protein. (B) Genome maps of all Ads constructed are shown. Ad vectors were designed as described in “Adenovirus vector construction: incorporation of DAF in the C-terminus of protein IX.” Capsid protein IX is outlined as Ad capsid protein used for fusion with DAF or DAF_REO. Letter “d” before DAF or GFP defines that this peptide is capsid-displayed. DAF orientation is depicted; arrowhead represents DAF C-terminus. Note that naturally DAF N-terminus is protruding from cell membrane (DAF_REO orientation). Genomes are not drawn to scale. ITR indicates inverted terminal repeat; and CMV, cytomegalovirus.