Persistent Growth of a Human Plasma-Derived Hepatitis C Virus Genotype 1b Isolate in Cell Culture

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics

A New Signaling Pathway for HCV Inhibition by Estrogen: GPR30 Activation Leads to Cleavage of Occludin by MMP-9.

Poor outcome in response to hepatitis C virus, including higher viral load, hepatocellular carcinoma and cirrhosis, is more associated with men and postmenopausal women than with premenopausal women and women receiving hormone replacement therapy, suggesting that β-estradiol plays an innate role in preventing viral infection and liver disease. Consequently, most research in the field has concluded that estrogen affects HCV replication through viral interactions with estrogen receptor-α. Previously, estrogen-like antagonists, including Tamoxifen, were shown to reduce HCV RNA production and prevent viral entry, although the authors did not identify host factors involved. Estrogen can act alternatively through the membrane-bound G-protein-coupled estrogen receptor, GPR30. Here, human hepatoma Huh7.5 cells were infected with HCV J6/JFH-1 and treated with estrogen or Tamoxifen, resulting in a marked decrease in detectable virus. The effect was mimicked by G1, a GPR30-specific agonist, and was reversed by the GPR30-specific antagonist, G15. While previous studies have demonstrated that estrogen down-regulated occludin in cervical cancer cells, its action on liver cells was unknown. Occludin is a tight junction protein and HCV receptor and here we report that activation and cellular export of MMP-9 led to the cleavage of occludin upon estrogen treatment of liver cells. This is the first report of the cleavage of an HCV receptor in response to estrogen. We also identify the occludin cleavage site in extracellular Domain D; the motif required for HCV entry and spread. This pathway gives new insight into a novel innate antiviral pathway and the suboptimal environment that estrogen provides for the proliferation of the virus. It may also explain the disparate host-virus responses to HCV demonstrated by the two sexes. Moreover, these data suggest that hormone replacement therapy may have beneficial antiviral enhancement properties for HCV-infected postmenopausal women and show promise for new antiviral treatments for both men and women

HCV-Mediated Apoptosis of Hepatocytes in Culture and Viral Pathogenesis.

Chronic Hepatitis C Virus (HCV) infection is associated with progressive liver injury and subsequent development of fibrosis and cirrhosis. The death of hepatocytes results in the release of cytokines that induce inflammatory and fibrotic responses. The mechanism of liver damage is still under investigation but both apoptosis and immune-mediated processes may play roles. By observing the changes in gene expression patterns in HCV-infected cells, both markers and the causes of HCV-associated liver injury may be elucidated. HCV genotype 1b virus from persistently infected VeroE6 cells induced a strong cytopathic effect when used to infect Huh7.5 hepatoma cells. To determine if this cytopathic effect was a result of apoptosis, ultrastructural changes were observed by electron microscopy and markers of programmed cell death were surveyed. Screening of a human PCR array demonstrated a gene expression profile that contained upregulated markers of apoptosis, including tumor necrosis factor, caspases and caspase activators, Fas, Bcl2-interacting killer (BIK) and tumor suppressor protein, p53, as a result of HCV genotype 1b infection. The genes identified in this study should provide new insights into understanding viral pathogenesis in liver cells and may possibly help to identify novel antiviral and antifibrotic targets

Schematic of the E2 signaling pathway controlling HCV entry and spread in liver cells.

<p>The nuclear receptor mediated pathway is activated by E2 and blocked by Tamoxifen (Tam) in most cell types. Activation of GPR30 is inhibited by G15 and initiated by E2, Tam or G1, leading to activation/secretion of MMP-9, which cleaves occludin in extracellular Domain D, resulting in a defective HCV receptor.</p

Estrogen abrogates HCV infection.

<p>(A) Huh7.5 cells respond to estrogen treatment measured by cyclin-D1 expression. (B) HCV core protein in J6/JFH1-infected Huh7.5 cells treated with E2 or Universal Type I IFN. (C) HCV intracellular core protein in J6/JFH1-infected Huh7.5 cells treated with E2, as measured by Flow Cytometry. (D) HCV RNA quantitated relative to GAPDH RNA levels from infected Huh7.5 cells treated with E2 or IFN. (E) Virus growth measured by absolute mean focus forming units (FFU). Error bars indicate the statistical standard deviation from the mean (±SD). Statistical significance is indicated by asterisks where: (*** = P ≤ 0.001; **** = P ≤ 0.0001). (F) Representative images of foci after 48 hr of E2 treatment. Magnification 40X (<i>below</i>); 100X (<i>above</i>).</p

E2 modulates matrix metalloproteinase to inhibit HCV growth.

<p>(<i>A</i>) Response of increasing concentrations of the broad-spectrum MMP inhibitor, ONO-4817 on J6/JFH1-infected Huh7.5 cells, in the presence or absence of E2 (<i>center panel</i>), and G1 (<i>right panel</i>) on HCV growth. Increasing concentrations of the broad spectrum MMP inhibitors (<i>B</i>) CP471,474 (C) Ilomastat (D) MMP408 or (E) SB-3CT. Data represent the absolute mean foci number of three independent experiments and error bars represent the standard deviation from the mean (±SD). Statistical significance is expressed as asterisks where; (* = P≤ 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001).</p

MMP-9 activation mediated by E2 modulates occludin.

<p>(A) Immunoblot of occludin down-regulation by G1, reversed by ONO-4817 (ONO). Protein expression relative to GAPDH was quantitated in a SyngeneG:box (<i>right</i>). (B) Immunoblot of full-length and truncated forms of occludin in Huh7.5 cells treated with G1 and ONO-4817 and the percentage of occludin forms were calculated relative to GAPDH protein levels (values below each band). (C) Schematic representation of occludin (504 amino acids). (D) Immunoblot of pro-MMP-9 (92 KDa) and active MMP-9 (~64 KDa) in Huh7.5 cell membranes and conditioned media (extracellular) following E2 treatment. (E) Immunoblot of MMP-9 in supernatant (extracellular) after siRNA transfection and E2 treatment (0, 1, 100, 10,000 nM). Cleaved occludin (38 KDa) is shown and GAPDH is shown as a gel loading control.</p

Effect of E2 on occludin and claudin-1.

<p>(A) Immunoblots of occludin and (B) claudin-1 protein from Huh7.5 cells treated with 10 μM E2. (C) Expression of HCV receptors occludin, claudin-1 and SR-B1 in Huh7.5 cells treated with increasing amounts of E2 or (D) G1. Intensity of immunoblot bands was quantified relative to GAPDH protein levels (<i>right</i>).</p