The serpin-secretion profile in Huh-7.5.1 cells was examined by Western blotting of cell lysates and extracellular media. As expected, a prominent 45-kDa band was detected in Ad-Spn4A.RRLL(r)-infected and Ad-Spn4A.RRLL(s)-infected cell lysates using anti-FLAG antibody (Figure 2A, lanes 2 and 3) . Furthermore, as hypothesized, the 45-kDa band of Spn4A.RRLL(r) was found only in lysed cell extracts and was not found secreted into extracellular media (Figure 2A Serpin-like properties and intrinsic specificity of Spn4A.RRLL(r) and Spn4A.RRLL(s) A stable, acyl-enzyme complex is formed between a protease and a functional inhibitory serpin following RCL cleavage. This allows for detection of the high molecular weight, heat-stable, and SDS-stable enzyme-inhibitor (EI) complex by standard SDS- Spn4A.RRKR(r) encodes for the naturally occurring serpin Spn4A, isolated from Drosophila melanogaster, with potent inhibitory activity against the human proprotein convertase furin. Spn4A.RRKR(r) contains the alpha-1 antitrypsin signal peptide (SP) at the N-terminus followed by a tandem His-tag (HHHHHH) and FLAG-tag (DYKDDDDK) sequence (HF). The P4 -P1 furin cleavage sequence in the RCL is Arg-Arg-Lys-Arg. Spn4A.RRKR(r) also contains the His-Asp-Glu-Leu (HDEL) ER retention motif (r) at the C-terminus. The secreted (s) serpin, Spn4A.RRKR(s), contains a stop codon before the HDEL signal. The RCL of Spn4A-RRKR(r) and (s) was modified to mimic the predicted SKI-1/S1P target cleavage site present in the Lassa virus glycoprotein pre-GP-C, which is Arg-Arg-Leu-Leu. Thus, Spn4A.RRLL(r), which is also retained in the ER, encodes the P4 -P1 Arg-Arg-Leu-Leu cleavage recognition sequence in the RCL. Spn4A.RRLL(s) contains a stop codon before HDEL, allowing the serpin to be secreted. (B) In silico homology model of the Spn4A.RRLL(r) variant was generated as described in the Materials and Methods. Ribbon diagram of the molecular model was generated using Pymol. The side chains of the RRLL residues within the flexible ''bait region'' of the RCL are shown as sticks in wheat colour. Sheet A is shown in yellow, sheet B is in blue, and sheet C is in cyan. Alpha-helices are red and loops are green. doi:10.1371/journal.ppat.1002468.g001 PAGE and Western blot [54, 55, 58, 62] . To determine if Spn4A.RRLL(s) is a functional and selective inhibitor of SKI-1/ S1P, recombinant Spn4A variants (furin-and SKI-1/S1P-directed inhibitors) were expressed in Huh-7.5.1 cells for 72 hours. Cell media and extracts containing recombinant serpins were then harvested and incubated with purified recombinant His-tagged human SKI-1/S1P or furin ( Figure 2B ) as described previously [47, 54, 58] . Reaction mixtures were analyzed by Western blot and probed for EI complex formation with anti-His antibody ( Figure 2B ) and anti-FLAG antibody ( Figure S3 ). The results shown in Figure 2B clearly demonstrate EI complex formation between recombinant SKI-1/S1P and Spn4A.RRLL(s) in cell media and in cell extracts (lane 5, upper and lower panels). As expected, Spn4A.RRLL(s) did not form a complex with furin ( Figure 2B , lane 10, upper and lower panels), whereas the furindirected serpin Spn4A.RRKR(s) formed an EI complex with furin but not with SKI-1/S1P in cell media and extracts ( Figure 2B , lanes 9 and 7, respectively, upper and lower panels). Lysed cellular extracts expressing Spn4A.RRLL(r) also demonstrated EI complex formation with SKI-1/S1P ( Figure 2B , lane 12, bottom panel). The results of our biochemical analysis confirmed the serpin-like properties of recombinant Spn4A.RRLL(r) and (s) biosynthesized in human hepatoma cells and the selectivity of Spn4A.RRLL(s) against SKI-1/S1P. Importantly, Spn4A.RRLL(s) inhibits SKI-1/ S1P by a suicide substrate mechanism and forms a kinetically trapped heat-and SDS-stable complex with SKI-1/S1P as is characteristic of other physiological serpin-protease pairs [58, 63] .