Figure 1.
Engineering the Spn4A scaffold to target the human subtilase SKI-1/S1P.
(A) Spn4A variants and amino-acid sequences of the engineered reactive center loop (RCL) “bait” region are shown. 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.
Figure 2.
Cellular expression and serpin-like properties of recombinant adenovirus-expressed Spn4A variants.
(A) The cellular expression of serpin variants Spn4A.RRLL(r) and (s) were examined using infectious adenoviral-mediated expression (moi 50) in Huh-7.5.1 cells. An empty adenovirus vector (Ad-Empty) was used as a control. After 48 hours, cell media (upper panel) and lysates (lower panels) were subjected to Western blot analysis. Spn4A variants were detected with mouse anti-FLAG antibody and, to ensure equal loading of samples, extracts were also probed with rabbit anti-β-tubulin antibody. (B) Huh-7.5.1 cells were infected with recombinant adenovirus expressing the His- and FLAG-tagged Spn4A variants indicated or the Ad-Empty control for 72 hours. Media alone (upper panels) or cell extracts (lower panels) lysed in RIPA buffer were combined with recombinant His-tagged SKI-1/S1P or His-tagged furin for 30 minutes at 30°C. Samples were prepared for Western blot analysis and probed with mouse anti-His antibody to detect SDS- and heat-stable protease-serpin complex formation as described in the Materials and Methods. Representative Western blots of at least 2 individual experiments are shown.
Figure 3.
PF-429242 and Spn4A.RRLL(s) inhibit SREBP activation and reduce neutral lipid abundance in Huh-7.5.1 cells.
(A) Huh-7.5.1 cells were treated with DMSO or 10 µM PF-429242 for 24 hours or infected with Ad-Empty (control), Ad-Spn4A.RRLL(r) or Ad-Spn4A.RRLL(s) for 48 hours. Cell extracts were harvested and subjected to nuclear fractionation as described in the Materials and Methods. Nuclear extracts were probed for N-terminal SREBP-1 expression and for enrichment of the nucleolar marker, fibrillarin. The * denotes a non-specific band detected by polyclonal anti-SREBP-1 antibody. (B) Huh-7.5.1 cells were grown in LPDS-supplemented media for 24 hours and were then infected with Ad-Spn4A.RRLL(r), Ad-Spn4A.RRLL(s), or Ad-Empty (control) for 72 hours; cell extracts were harvested and subjected to Western blot analysis. Anti-PCSK9, LDLR, SREBP-2, and GM130 antibodies were used to detect protein expression levels, and β-tubulin was probed for normalizing band intensities. Values are plotted relative to protein expression in control cells (left panel). The right panel shows representative Western blots for the effect of serpin on each protein examined. (C) Cells were treated with DMSO (control) or 10 µM PF-429242 for 24 hours. The compound was removed and the cells incubated for an additional 24 hours. Cells were then harvested and the relative levels of intracellular lipids were determined in treated cells relative to control cells. (D) Huh-7.5.1 cells were infected with Ad-Spn4A.RRLL(r), Ad-Spn4A.RRLL(s), or Ad-Empty (control) for 72 hours. Cells were then harvested and the relative levels of intracellular lipids were determined in treated cells relative to the control. In (A), a representative Western blot of 3 independent experiments is shown. For (B), (C), and (D), results (mean ± SEM) from 3 independent experiments are shown. *p<0.05; **p<0.01; ***p<0.005.
Figure 4.
LD abundance is dramatically reduced in Huh-7.5.1 cells expressing Spn4A.RRLL(s).
(A-C) Huh-7.5.1 cells were infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 72 hours. Fixed cells were stained for cell nuclei using Hoechst dye (blue) and probed for expression of Spn4A variants using mouse anti-FLAG antibody (red). (A) LDs were detected using BODIPY 493/503 (green), and images were acquired using a Leica TCSSP5 confocal microscope. (B) MetaMorph imaging software was used to quantify the number of BODIPY-stained LDs in control cells (n = 23) and individual cells expressing Spn4A.RRLL(r) (n = 21) or Spn4A.RRLL(s) (n = 15). Acquisition and analysis were performed using the same intensity and threshold settings across all images. (C) The LD marker ADRP was detected in cells treated with Spn4A.RRLL(r), Spn4A.RRLL(s), and Ad-Empty (control) using rabbit anti-ADRP antibody (green), and images were obtained using an Olympus Fluoview FV1000 laser scanning confocal microscope. (D) Huh-7.5.1 cells infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 72 hours were harvested and subjected to SDS-PAGE and Western blot analysis. Mouse anti-ADRP antibody was used to detect protein expression levels in serpin-treated cells compared to control-treated cells. Relative protein expression was quantified by normalizing to β-tubulin expression. The inset shows a representative Western blot. (E) Huh-7.5.1 cells were treated with DMSO (control) or with 10 µM PF-429242 for 24 hours, the compound was removed, and the cell lysates were harvested after an additional 48 hours. Relative ADRP expression (normalized to β-tubulin) in inhibitor-treated cells compared to control cells was quantified by subjecting total cell lysates to Western blot analysis. Values are plotted relative to protein expression in control cells, which are set to 1. Results (mean ± SEM) from 3 independent experiments are shown. *p<0.05.
Figure 5.
Inhibition of SKI-1/S1P using Spn4A.RRLL(s) results in a dose-dependent inhibition of HCV infection in Huh-7.5.1 cells.
(A) Huh-7.5.1 cells were infected with moi (1 – 50) of Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 48 hours in regular media or media supplemented with sterols. Treated cells were infected with HCV (moi 0.1) and fixed 72 hours post-HCV-infection. Cells, probed with HCV anti-core antibody (red) and stained with Hoechst dye (nuclei; blue), were quantified using Cellomics HCS to determine the percentage of HCV-infected cells. Sample images of cells infected with adenovirus moi 50 acquired with Cellomics HCS using the 10 X objective are shown below the graph. (B) Huh-7.5.1 cells were infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) (moi 50) for 48 hours in complete media and then infected with HCV (moi 0.1) for 72 hours. Relative HCV-core expression (normalized to β-tubulin) in serpin-treated cells compared to control-treated cells was quantified by examining total cell lysates using Western blot analysis. Infectious HCV titer in the extracellular media was also determined and relatively quantified. (C) Control and serpin-treated cells were infected with HCV or transfected with genomic HCV RNA, and total cellular RNA was harvested 72 hours later. HCV RNA levels, normalized to β-actin transcript levels, were relatively quantified in cell extracts using real-time PCR. (D) Huh.2 and Huh.8 replicon-harbouring cells were treated with serpin-expressing and control adenoviruses for 5 days before total RNA was harvested. HCV RNA levels, normalized to β-actin transcript levels, were relatively quantified in cell extracts using real-time PCR. All values are expressed as relative HCV levels in serpin-treated cells compared to control-treated cells. Results (mean ± SEM) from at least 3 independent experiments are shown. Statistical significance was calculated for Ad-Spn4A.RRLL(r) or Ad-Spn4A.RRLL(s) infection compared to Ad-Empty infection at the same moi. *p<0.05; **p<0.01; ***p<0.005.
Figure 6.
Inhibition of SKI-1/S1P using PF-429242 results in a dose-dependent inhibition of HCV infection, pre- and post-establishment of viral infection in Huh-7.5.1 cells.
(A) Huh-7.5.1 cells were treated with various concentrations (0.01 to 50 µM) of PF-429242 for 24 hours. The inhibitor was removed and the cells were then infected with HCV (moi 0.1) in complete media for 48 hours. Cells, probed with HCV anti-core antibody (red) and stained with Hoechst dye (nuclei; blue), were quantified using Cellomics HCS to determine the percentage of HCV-infected cells. The average EC50 value from 3 independent experiments is displayed above the graph. Sample images of cells treated with DMSO or 50 µM PF-429242 acquired with Cellomics HCS using the 10 X objective are shown to the left of the graph. (B) Cells were treated with different concentrations (0.1 to 40 µM) of PF-429242 either 24 hours before (left half of graph) or 24 hours after (right half of graph) infection with HCV (moi 0.1). At 72 hours post-HCV infection, cells were fixed and probed with HCV anti-core antibody (red) and stained with Hoechst dye (nuclei; blue) to determine the percentage of HCV-infected cells (core) using Cellomics HCS. Also, media from treated and HCV-infected cells was harvested and the amount of extracellular, infectious HCV was titered (HCV titer; FFU/ml). Results (mean ± SEM) from 3 independent experiments are shown. (C) Huh.8 and Huh.2 replicon-harbouring cells were treated with DMSO (control) or 10 µM PF-429242 for 72 hours before total RNA levels were harvested. HCV RNA levels, normalized to β-actin transcript levels, were relatively quantified in cell extracts using real-time PCR. Results (mean ± SEM) from 2 independent experiments are shown. Statistical significance was calculated for PF-429242 treated cells compared to DMSO-treated cells. *p<0.05; **p<0.01; ***p<0.005.
Figure 7.
SKI-1/S1P is a novel potential target for indirect-acting antiviral agents against HCV infection.
(1) The inactive SKI-1/S1P zymogen is biosynthesized in the ER and traffics to the Golgi apparatus following intramolecular autocatalytic maturation of the proenzyme [35], [46], [99], [100]. (2) During HCV infection, the SREBP pathway is activated by a variety of molecular mechanisms [19], [30]–[32]. (3) For SREBP to activate genes involved in lipid biosynthesis, its N-terminal domain must be released through sequential endoproteolytic cleavage first by SKI-1/S1P and then by S2P [37], [38]. (4) The released N-terminal domain translocates to the nucleus and activates various aspects of lipid metabolism [36]. (5) Activation of lipid biosynthesis increases LD formation where the HCV core protein localizes to orchestrate HCV assembly and subsequent secretion [8], [10], [16]. (6) Biosynthesis of LDLR, a proposed receptor for HCV entry, is also activated by SREBP signaling [69], [79], [101]. (7) Spn4A.RRLL(s) is a secretory pathway-expressed serpin (Figure 1). (8) Spn4A.RRLL(s) interacts and forms a covalent complex with enzymatically active SKI-1/S1P molecules (Figure 2 and S3) in the Golgi apparatus preventing SKI-1/S1P-mediated endoproteolytic cleavage of SREBP protein (Figure 3A). (9) A small-molecule inhibitor PF-429242 also efficiently inhibits SKI-1/S1P endoproteolytic activity (Figure 3A). (10) SKI-1/S1P inhibition blocks expression of the putative HCV receptor, LDLR (Figure 3B and S4), and reduces HCV entry (Figure 5). (11) The expression of other SREBP-regulated genes, such as PCSK9 and SREBP-2, are also blocked (Figure 3B). (12) Downstream lipid synthesis is interrupted resulting in overall reduced intracellular cholesterol-ester and triglyceride abundance (Figure 3C and 3D). (12) This is then detected as a decrease in LD abundance (Figure 4), which impedes assembly and secretion of infectious HCV particles.