A Serine Palmitoyltransferase Inhibitor Blocks Hepatitis C Virus Replication in Human Hepatocytes

Background & AimsHost cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice.MethodsWe tested the ability of NA808 to inhibit SPT’s enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors.ResultsNA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors.ConclusionsThe SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

<div><p>Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (<em>SGMS1</em> and <em>2</em>) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.</p> </div

Relationship between the SGMS genes and HCV infection.

<p>(<b>A, B</b>) The correlation between SGMS1/2 and liver HCV-RNA of HCV infected humanized chimeric mice (n = 7). (<b>C</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on HCV in HCV-infected cells. (<b>D</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on the expression of SGMS1/2 mRNA measured by RTD-PCR. (<b>E</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM each) measured by RTD-PCR. (<b>F</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM) on HCV replication in FLR 3-1. In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01.</p

Effects of NA808 treatment on sphingomyelin (SM) and ceramide (total and individual molecular species).

<p>(<b>A, B</b>) Relative ratio of total ceramide (<b>A</b>) and SM (<b>B</b>) in uninfected mice (white, n = 4), HCV genotype 1a-infected mice (black, n = 5), and HCV-infected mice treated with NA808 for 14 days (dark gray, 5 mg/kg, n = 4; light gray, 10 mg/kg, n = 3). *<i>p</i><0.05 and **<i>p</i><0.01 compared with HCV-infected mice. (<b>C</b>) SM levels (bars) and HCV RNA levels (black arrowhead) in the livers of mice treated for 14 days with NA808 (5 or 10 mg/kg/day) and untreated chimeric mice. (<b>D, E</b>) Relative intensities of individual ceramide molecular species (<b>D</b>) and individual SM molecular species (<b>E</b>) in uninfected mice (white, n = 3), HCV-infected mice (black, n = 3), and HCV-infected mice treated with NA808 for 14 days (dark gray, 5 mg/kg, n = 2; light gray, 10 mg/kg, n = 1). In all cases, error bars indicate SDs.</p

HCV alters sphingolipid metabolism.

<p>(<b>A, B</b>) Time-course studies of humanized chimeric mice inoculated with human serum samples positive for HCV genotype 1a (<b>A</b>) or 2a (<b>B</b>). (<b>C</b>) mRNA expression of <i>SGMS1</i> and <i>SGMS2</i> in uninfected (white, n = 5) and HCV genotype 1a-infected (black, n = 7) chimeric mice. (<b>D, E</b>) Effects of HCV infection on hepatocyte SM and ceramide levels in humanized chimeric mice. Relative intensity of total ceramide (<b>D</b>) and total shingomyelin (SM) (<b>E</b>) in uninfected mouse hepatocytes (white bar, n = 4), HCV genotype 1a-infected mouse hepatocytes (black bar, n = 5), and HCV genotype 2a-infected mouse hepatocytes (dark gray bar, n = 3). (<b>F</b>) Mass spectrum of SM in Bligh & Dyer extracts of a human hepatocyte cell line (HuH-7 K4). (<b>G, H</b>) Effects of HCV infection on hepatocyte SM and ceramide levels in humanized chimeric mice. Relative intensity of individual ceramide molecular species (<b>G</b>) and individual SM molecular species (<b>H</b>) in uninfected mouse hepatocytes (white bar, n = 3), HCV genotype 1a-infected mouse hepatocytes (black bar, n = 3), and HCV genotype 2a-infected mouse hepatocytes (dark gray bar, n = 3). In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01 compared with uninfected hepatocytes.</p

Characterization of the hepatotropic serine palmitoyltransferase inhibitor NA808.

<p>(<b>A</b>) Sphingolipid biosynthesis pathway and structure of NA808. (<b>B</b>) Activity of SPT in FLR3-1 cells after 72 h of NA808 treatment. **<i>p</i><0.01 compared with control. (<b>C</b>) Results of TLC showing <i>de novo</i> sphingolipid biosynthesis in the presence of NA808. Cer = ceramide, PE = phosphatidylethanolamine, PC = phosphatidylcholine, SM = sphingomyelin. (<b>D</b>) Immunosuppressive activity of NA808. Cyclosporin A (CsA) and tacrolimus (FK-506) were used as positive controls. (<b>E</b>) Effects of NA808 on HCV replication (black bars) and cell viability (gray symbols) in FLR 3-1 replicon-containing cells. Error bars indicate SDs. (<b>F</b>) Effects of NA808 on the level of the RdRp and β-actin, as assessed by Western blotting. (<b>G</b>) Effect of NA808 on the production of HCV NS3 protein (green) in FLR3-1 replicon-containing cells, as assessed by immunofluorescence analysis. Nuclear DNA was stained with DAPI (blue).</p

Inhibition of sphingolipid biosynthesis with hepatotropic serine palmitoyltransferase (SPT) inhibitor NA808 exerts anti-HCV effect.

<p>(<b>A</b>) Serum HCV-RNA levels in response to treatment with NA808 (blue, 5 mg/kg/day, purple, 10 mg/kg/day, n = 6 each), or PegIFN-α (pink, 30 µg/kg twice weekly, n = 4). (<b>B</b>) Effect of NA808 (5 mg/kg/day) on serum HCV-RNA levels. A star indicates that HCV-RNA was not detected. (<b>C</b>) Levels of liver HCV-RNA (black) and HCV core protein (gray) after the 14-day treatment. *<i>p</i><0.05 and **<i>p</i><0.01 compared with no treatment. (<b>D</b>) Histological analysis using immunofluorescent labeling of HCV core protein (green) and fluorescent staining of nuclei (blue). (<b>E</b>) Serum HCV-RNA levels in response to no treatment (pink, n = 3) or NA808 treatment (blue, 5 mg/kg/day, n = 4). (<b>F</b>) Liver HCV-RNA levels in genotype 2a-infected mice after the 14-day treatment. *<i>p</i><0.05 and **<i>p</i><0.01 compared with no treatment. In all cases, error bars indicate SDs.</p

Specific sphingomyelin molecular species upregulated by HCV promote HCV replication on the detergent-resistant membrane fraction.

<p>(<b>A</b>) Comparison of the relative amounts of SM, as measured by MS analysis, in whole cells and the DRM fraction of mock-infected (HuH-7 K4 cells) (white, n = 6; whole cells, n = 3; DRM fraction) and HCV (JFH-1)-infected cells (JFH/K4 cells) (black, n = 6; whole cells, n = 3; DRM fraction). (<b>B</b>) Composition ratio of SM molecular species in whole cells and DRM fraction of HCV-infected cells. (<b>C</b>) Relative intensities of each SM molecular species in the DRM fraction of mock-infected cells (white, n = 2) and HCV-producing cells without (black, n = 2) or with NA808 treatment (gray, n = 2). (<b>D</b>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002860#s2" target="_blank">Results</a> of the ELISA SM binding assay (n = 3 each). (<b>E</b>) Average activation kinetics of each SM molecular species on HCR6 (genotype 1b) RdRp (n = 3 each). (<b>F</b>) Scheme of HCV-RNA replicase assay using digitonin-permeabilized cells. (<b>G, H</b>) Effect of each SM molecular species on HCV-RNA in digitonin-permeabilized replicon cells treated without (G) or with 10 nM NA808 (H) (n = 3 each). In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01.</p