Elsevier

Antiviral Research

Volume 115, March 2015, Pages 94-104
Antiviral Research

SKI-1/S1P inhibitor PF-429242 impairs the onset of HCV infection

https://doi.org/10.1016/j.antiviral.2014.12.017Get rights and content

Highlights

  • SKI-1/S1P small molecule inhibitor PF-429242 inhibits HCV entry.

  • PF-429242 inhibits HCV entry in SREBP-dependent and independent manner.

  • SREBP-dependent inhibition of HCV viral entry relies on LDLR and NPC1L1.

Abstract

Worldwide, approximately 170 million individuals are afflicted with chronic hepatitis C virus (HCV) infection. To prevent the development of inherent diseases such as cirrhosis and hepatocellular carcinoma, tremendous efforts have been made, leading to the development of promising new treatments. However, their efficiency is still dependent on the viral genotype. Additionally, these treatments that target the virus directly can trigger the emergence of resistant variants. In a previous study, we have demonstrated that a long-term (72 h) inhibition of SKI-1/S1P, a master lipogenic pathway regulator through activation of SREBP, resulted in impaired HCV genome replication and infectious virion secretion. In the present study, we sought to investigate the antiviral effect of the SKI-1/S1P small molecule inhibitor PF-429242 at the early steps of the HCV lifecycle. Our results indicate a very potent antiviral effect of the inhibitor early in the viral lifecycle and that the overall action of the compound relies on two different contributions. The first one is SREBP/SKI-1/S1P dependent and involves LDLR and NPC1L1 proteins, while the second one is SREBP independent. Overall, our study confirms that SKI-1/S1P is a relevant target to impair HCV infection and that PF-429242 could be a promising candidate in the field of HCV infection treatment.

Introduction

The hepatitis C virus (HCV) chronically infects 170 million individuals worldwide. HCV persistence often triggers liver steatosis, cirrhosis, and hepatocellular carcinoma (HCC) (Di Bisceglie, 1997). Tremendous improvement has been achieved in the treatment of chronically infected patients (deLemos and Chung, 2014) with sustained viral response up to 98%. However, treatment efficiencies depend on the genotype and can in some cases trigger the appearance of adaptive variants. Recently developed molecules are direct-acting antivirals (DAA) that target either the protease complex NS3/4A, the multifunctional NS5A protein, or the viral polymerase NS5B.

Since antiviral compounds that target cellular factors result in less drug resistant variants than those targeting viral proteins (Provencher et al., 2004), we have previously studied the effect of the inhibition of subtilisin/hexin-isoenzyme-1/site-1 protease (SKI-1/S1P) on HCV propagation. SKI-1/S1P belongs to the proprotein convertase family (Seidah and Prat, 2007) and is known to be a master regulator of cellular lipid homeostasis (Horton et al., 2002), a process on which HCV tightly relies (Alvisi et al., 2011). We have shown that PF-429242, a cell permeable SKI-1/S1P inhibitor [developed by Pfizer (Hawkins et al., 2008)], when added to the cells for 72 h, could decrease HCV genome replication and infectious particle production (Blanchet et al., 2012). A recent study suggested that SKI-1/S1P could additionally have a role at the entry level (Olmstead et al., 2012), but data supporting this observation are still missing.

SKI-1/S1P regulates genes involved in cholesterol and fatty acid synthesis and cellular intake. When the intracellular cholesterol concentration decreases, sterol responsive element binding proteins (SREBPs), located in an inactive precursor state at the endoplasmic reticulum (ER) membrane, are targeted to the Golgi. After initial cleavage by SKI-1/S1P in the cis/medial Golgi followed by a second cleavage by the membrane-bound metalloprotease S2P, the cytosolic amino-terminal active moiety of SREBP (NtSREBP) is released from the membrane and targeted to the nucleus, where it acts as a transcription factor to promote lipogenesis (Horton et al., 2002).

The HCV lifecycle is tightly dependant on lipid metabolism. Importantly, HCV virions mostly associate with very low density lipoprotein (VLDL) during morphogenesis and secretion to form lipoviroparticles (LVPs). This type of particle is assumed to be highly infectious (Andre et al., 2002, Boyer et al., 2014, Merz et al., 2011). Because of this association, HCV uptake is dependent on both viral and lipid specific receptors (Zeisel et al., 2013). Among the known HCV receptors, several have been suggested to be modulated in an SREBP-dependant manner (Fig. 1). LDLR and NPC1L1 promoters bear SRE sequences (Attie and Seidah, 2005, Kotzka et al., 2000, Li et al., 1999, Pramfalk et al., 2010). SR-B1 promoter contains E-box sequences (Cao et al., 1997), known to be somewhat activated by SREBP (Amemiya-Kudo et al., 2002). The presence of such motifs has not been documented for CD81. However PCSK9, a member of the proprotein convertase family, itself regulated in a SREBP/SKI-1/S1P-dependant manner (Dubuc et al., 2004, Li et al., 2009), has been shown to reduce CD81 cellular concentration through a yet unknown mechanism (Labonte et al., 2009). Importantly, PCSK9 is also capable of reducing the concentration of LDLR (Attie and Seidah, 2005, Seidah et al., 2014).

Given these complex regulations, we sought to examine the effect of SKI-1/S1P inhibition on the expression profile of HCV receptors mRNA and proteins, as well as on HCV ability to infect cells.

Section snippets

Cell culture and viruses

Huh7.5 and Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin. Cytotoxicity and cellular gene expressions tests were conducted with media containing 10% lipoprotein-deficient serum (LPDS-Biomedical Technologies). Other experiments, unless otherwise indicated, were performed with 10% FBS containing media. HCV infection assays were conducted using the JFH1 viral strain on Huh7.5 cell cultures as described

SKI-1/S1P inhibitor PF-429242 specifically inhibits SRE promoter-driven gene expression

To evaluate the propensity of PF-429242 to alter the synthesis of known HCV receptors mRNA, Huh7.5 cells were exposed to various concentrations of the SKI-1/S1P inhibitor for 24 h with no observable cellular toxicity (Fig. S1) and measured the outcome on HCV receptors mRNA concentrations (Fig. 2). LPDS-based media was initially used to improve the readout of the experiment. Indeed, reduction in extracellular lipid concentration results in an increased baseline of SREBP-dependant mRNA

Discussion

Chronic HCV infection can lead to severe liver diseases such as steatosis, cirrhosis and hepatocellular carcinoma. In a recent study we have reported that the inhibition of SKI-1/S1P, a master lipogenic regulator, could impair HCV genome replication, lipid droplet homeostasis and the production of infectious HCV virions (Blanchet et al., 2012).

In continuity with this first study, we sought to investigate the effect of the SKI-1/S1P inhibitor PF-429242 at the early steps of HCV lifecycle.

Acknowledgements

P.L. is supported by a Canadian Institutes of Health Research (CIHR) Grant #MOP 93792 and received a salary support from Le Fonds de recherche du Québec (FRSQ). M.B. was supported by a postdoctoral Fellowship from “La fondation Universitaire Armand-Frappier”. N.G.S. is supported by a CIHR Grant #MOP 93792 and by a Canada Research Chair #216684. C.S. is a CNRS investigator and is supported by a grant from the “Agence nationale de recherches sur le sida et les hépatites virales”. We would like to

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