Human Subtilase SKI-1/S1P Is a Master Regulator of the HCV Lifecycle and a Potential Host Cell Target for Developing Indirect-Acting Antiviral Agents

HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1) – or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care

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.

<p>(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. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.005.</p

LD abundance is dramatically reduced in Huh-7.5.1 cells expressing Spn4A.RRLL(s).

<p>(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. *<i>p</i><0.05.</p

SKI-1/S1P is a novel potential target for indirect-acting antiviral agents against HCV infection.

<p><b>(1)</b> The inactive SKI-1/S1P zymogen is biosynthesized in the ER and traffics to the Golgi apparatus following intramolecular autocatalytic maturation of the proenzyme <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Nohturfft1" target="_blank">[35]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Seidah1" target="_blank">[46]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Elagoz1" target="_blank">[99]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Seidah2" target="_blank">[100]</a>. <b>(2)</b> During HCV infection, the SREBP pathway is activated by a variety of molecular mechanisms <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Waris1" target="_blank">[19]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Park1" target="_blank">[30]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Kim2" target="_blank">[32]</a>. <b>(3)</b> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Sakai1" target="_blank">[37]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Rawson1" target="_blank">[38]</a>. <b>(4)</b> The released N-terminal domain translocates to the nucleus and activates various aspects of lipid metabolism <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Brown2" target="_blank">[36]</a>. <b>(5)</b> Activation of lipid biosynthesis increases LD formation where the HCV core protein localizes to orchestrate HCV assembly and subsequent secretion <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Miyanari1" target="_blank">[8]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Shavinskaya1" target="_blank">[10]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Boulant2" target="_blank">[16]</a>. <b>(6)</b> Biosynthesis of LDLR, a proposed receptor for HCV entry, is also activated by SREBP signaling <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Owen1" target="_blank">[69]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Agnello1" target="_blank">[79]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468-Wang1" target="_blank">[101]</a>. <b>(7)</b> Spn4A.RRLL(s) is a secretory pathway-expressed serpin (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g001" target="_blank">Figure 1</a>). <b>(8)</b> Spn4A.RRLL(s) interacts and forms a covalent complex with enzymatically active SKI-1/S1P molecules (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g002" target="_blank">Figure 2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat.1002468.s003" target="_blank">S3</a>) in the Golgi apparatus preventing SKI-1/S1P-mediated endoproteolytic cleavage of SREBP protein (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g003" target="_blank">Figure 3A</a>). <b>(9)</b> A small-molecule inhibitor PF-429242 also efficiently inhibits SKI-1/S1P endoproteolytic activity (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g003" target="_blank">Figure 3A</a>). <b>(10)</b> SKI-1/S1P inhibition blocks expression of the putative HCV receptor, LDLR (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g003" target="_blank">Figure 3B and S</a>4), and reduces HCV entry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g005" target="_blank">Figure 5</a>). <b>(11)</b> The expression of other SREBP-regulated genes, such as PCSK9 and SREBP-2, are also blocked (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g003" target="_blank">Figure 3B</a>). <b>(12)</b> Downstream lipid synthesis is interrupted resulting in overall reduced intracellular cholesterol-ester and triglyceride abundance (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g003" target="_blank">Figure 3C and 3D</a>). <b>(12)</b> This is then detected as a decrease in LD abundance (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#ppat-1002468-g004" target="_blank">Figure 4</a>), which impedes assembly and secretion of infectious HCV particles.</p

Cellular expression and serpin-like properties of recombinant adenovirus-expressed Spn4A variants.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#s4" target="_blank">Materials and Methods</a>. Representative Western blots of at least 2 individual experiments are shown.</p

PF-429242 and Spn4A.RRLL(s) inhibit SREBP activation and reduce neutral lipid abundance in Huh-7.5.1 cells.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002468#s4" target="_blank">Materials and Methods</a>. 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. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.005.</p