SKI-1/S1P inhibitor PF-429242 impairs the onset of HCV infection
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
References (52)
- et al.
Characterization of the hepatitis C virus RNA replication complex associated with lipid rafts
Virology
(2004) - et al.
Transcriptional activities of nuclear SREBP-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes
J. Lipid Res.
(2002) - et al.
Dual regulation of the LDL receptor – some clarity and new questions
Cell Metab.
(2005) - et al.
Cell entry of hepatitis C virus
Virology
(2006) - et al.
SKI-1/S1P inhibition: a promising surrogate to statins to block hepatitis C virus replication
Antiviral Res.
(2012) - et al.
Use of FDA approved therapeutics with hNTCP metabolic inhibitory properties to impair the HDV lifecycle
Antiviral Res.
(2014) - et al.
Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB
J. Biol. Chem.
(2008) - et al.
The association of hepatitis C virus glycoproteins with apolipoproteins e and B early in assembly is conserved in lipoviral particles
J. Biol. Chem.
(2014) - et al.
Structure and localization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control by steroidogenic factor 1
J. Biol. Chem.
(1997) - et al.
Hepatitis C treatment: an incipient therapeutic revolution
Trends Mol. Med.
(2014)
Establishment of a hepatitis C virus subgenomic replicon derived from human hepatocytes infected in vitro
Biochem. Biophys. Res. Commun.
Sterol regulatory element binding proteins (SREBP)-1a and SREBP-2 are linked to the MAP-kinase cascade
J. Lipid Res.
Induction of low density lipoprotein receptor (LDLR) transcription by oncostatin M is mediated by the extracellular signal-regulated kinase signaling pathway and the repeat 3 element of the LDLR promoter
J. Biol. Chem.
Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine
J. Biol. Chem.
Novel putative SREBP and LXR target genes identified by microarray analysis in liver of cholesterol-fed mice
J. Lipid Res.
Antagonism of secreted PCSK9 increases low density lipoprotein receptor expression in HepG2 cells
J. Biol. Chem.
Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome
J. Biol. Chem.
HNF1alpha and SREBP2 are important regulators of NPC1L1 in human liver
J. Lipid Res.
The role of scavenger receptor class B type I (SR-BI) in lipid trafficking. Defining the rules for lipid traders
Int. J. Biochem. Cell Biol.
Identification of FBL2 as a geranylgeranylated cellular protein required for hepatitis C virus RNA replication
Mol. Cell
Cholesterol-regulated translocation of NPC1L1 to the cell surface facilitates free cholesterol uptake
J. Biol. Chem.
Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation
J. Biol. Chem.
Role of low-density lipoprotein receptor in the hepatitis C virus life cycle
Hepatology
Hepatitis C virus and host cell lipids: an intimate connection
RNA Biol.
Characterization of low- and very-low-density hepatitis C virus RNA-containing particles
J. Virol.
Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes
J. Exp. Med.
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