Youhui Si

High-Throughput Profiling of Alpha Interferon- and Interleukin-28B-Regulated MicroRNAs and Identification of let-7s with Anti-Hepatitis C Virus Activity by Targeting IGF2BP1

ABSTRACT Hepatitis C virus (HCV) infection is a major cause of severe liver disease. Interferon (IFN)/ribavirin treatment remains the standard therapeutic regimen for HCV infection in most countries. IFN-stimulated genes are believed to contribute to antiviral effects. However, emerging evidence suggests that microRNAs (miRNAs), a class of noncoding small RNAs, are involved in the control of viral infection. Here, we systematically profiled the hepatocyte expression of a set of 750 miRNAs in response to alpha interferon (IFN-α) and interleukin-28B (IL-28B) treatments. The anti-HCV activity of differentially expressed miRNAs was evaluated using cell culture-derived HCV in vitro. The results demonstrate that let-7b had a significant anti-HCV effect by inhibiting HCV replication and viral protein translation in human hepatoma cells. In particular, we show that the inhibition of let-7b attenuated the anti-HCV effects of IFN-α and IL-28B. Furthermore, we show that the host factor insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a target of let-7b. IGF2BP1 was required for HCV replication, and its expression was downregulated by IFN-α and IL-28B. Deletion of the wild-type seed region of let-7b abolished its antiviral activity. Finally, we demonstrate that other let-7 family miRNAs were able to inhibit HCV and to suppress IGF2BP1 expression. In conclusion, we provide an example of a host miRNA regulated by type I and type III IFNs that inhibits HCV replication and infectivity by targeting host targets. These results highlight the important role of miRNAs in the host antiviral immune response and provide a novel candidate for anti-HCV therapy.

A human claudin-1-derived peptide inhibits hepatitis C virus entry.

Hepatitis C virus (HCV) entry is a complicated process that requires multiple host factors, such as CD81, scavenger receptor BI, claudin‐1 (CLDN1), and occludin. The interaction of virus and cellular entry factors represents a promising target for novel anti‐HCV drug development. In this study, we sought to identify peptide inhibitors for HCV entry by screening a library of overlapping peptides covering the four above‐mentioned entry factors. An 18–amino acid peptide (designated as CL58) that was derived from the CLDN1 intracellular and first transmembrane region inhibited both de novo and established HCV infection in vitro. Unlike previously reported peptides corresponding to CLDN1 extracellular loops, CL58 did not alter the normal distribution of CLDN1 and was not cytotoxic in vitro at concentrations nearly 100‐fold higher than the effective antiviral dose. The inhibitory effect of CL58 appeared to occur at a late step during viral entry, presumably after initial binding. Finally, overexpressed CL58 was able to interact with HCV envelope proteins. Conclusion: We identified a novel CLDN1‐derived peptide that inhibits HCV entry at a postbinding step. The findings expand our knowledge of the roles that CLDN1 play in HCV entry and highlight the potential for developing a new class of inhibitors targeting the viral entry process. (HEPATOLOGY 2012)

Association of serum level of growth differentiation factor 15 with liver cirrhosis and hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) and liver cirrhosis are associated with high mortality worldwide. Currently, alpha-fetoprotein (AFP) is used as a standard serum marker for the detection of HCC, but its sensitivity and specificity are unsatisfactory, and optimal diagnostic markers for cirrhosis are lacking. We previously reported that growth differentiation factor 15 (GDF15) was significantly induced in HCV-infected hepatocytes. This study aimed to investigate GDF15 expression and its correlation with hepatitis virus-related liver diseases. A total of 412 patients with various liver diseases were studied. Healthy and Mycobacterium tuberculosis-infected subjects were included as controls. Serum and tissue GDF15 levels were measured. Serum GDF15 levels were significantly increased in patients with HCC (6.66±0.67 ng/mL, p<0.0001) and cirrhosis (6.51±1.47 ng/mL, p<0.0001) compared with healthy controls (0.31±0.01 ng/mL), though the GDF15 levels in HBV and HCV carriers were moderately elevated (1.34±0.19 ng/mL and 2.13±0.53 ng/mL, respectively). Compared with HBV or HCV carriers, GDF15 had a sensitivity of 63.1% and a specificity of 86.6% at the optimal cut-off point of 2.463 ng/mL in patients with liver cirrhosis or HCC. In HCC patients, the area under the receiver operating curve was 0.84 for GDF15 and 0.76 for AFP, but 0.91 for the combined GDF15 and AFP. Serum GDF15 levels did not significantly differ between the high-AFP and low-AFP groups. GDF15 protein expression in HCC was significantly higher than that in the corresponding adjacent paracarcinomatous tissue and normal liver. Using a combination of GDF15 and AFP will improve the sensitivity and specificity of HCC diagnosis. Further research and the clinical implementation of serum GDF15 measurement as a biomarker for HCC and cirrhosis are recommended.

Growth Differentiation Factor 15 Is Induced by Hepatitis C Virus Infection and Regulates Hepatocellular Carcinoma-Related Genes

Liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) are commonly induced by chronic hepatitis C virus (HCV) infection. We aimed to identify and characterize the involvement of previously screened cytokine GDF15 in HCV pathogenesis. We examined the GDF15 expression after HCV infection both in vitro and in vivo. Cultured JFH-1 HCV was used to determine the GDF15 function on virus propagation. GDF15 overexpression and RNA interference were employed to profile the GDF15-regulated genes, signaling pathways and cell biology phenotypes. The mRNA expression and protein secretion of GDF15 was dramatically increased in HCV-infected hepatoma cells, which maybe a host response to viral proteins or infection-induced cell stress. Patients infected with HCV had an average 15-fold higher blood GDF15 level than that of healthy volunteers. Three HCC individuals in the HCV cohort showed extremely high GDF15 concentrations. Transfection or exogenously supplied GDF15 enhanced HCV propagation, whereas knockdown of endogenous GDF15 resulted in inhibition of virus replication. Overexpressed GDF15 led to Akt activation and the phosphorylation of Akt downstream targeted GSK-3β and Raf. Several HCC-related molecules, such as E-cadherin, β-catenin, Cyclin A2/B1/D1, were up-regulated by GDF15 stimulation in vitro. Overexpression of GDF15 in hepatoma cells resulted in increased DNA synthesis, promoted cell proliferation, and importantly enhanced invasiveness of the cells. In conclusion, these results suggest that an elevated serum GDF15 level is a potential diagnostic marker for viral hepatitis, and GDF15 may contribute to HCV pathogenesis by altering the signaling and growth of host cells.

Phospholipid scramblase 1 mediates hepatitis C virus entry into host cells

E1 physically interacts with PLSCR1 by pull down (View interaction)

Screening and Rational Design of Hepatitis C Virus Entry Inhibitory Peptides Derived from GB Virus A NS5A

ABSTRACT Chronic infection by hepatitis C virus (HCV) is a cause of the global burden of liver diseases. HCV entry into hepatocytes is a complicated and multistep process that represents a promising target for antiviral intervention. The recently reported amphipathic α-helical virucidal peptide (C5A) from the HCV NS5A protein suggests a new category of antiviral drug candidates. In this study, to identify C5A-like HCV inhibitors, synthetic peptides derived from the C5A-corresponding NS5 protein region of selected Flaviviridae viruses were evaluated for their anti-HCV activities. A peptide from GB virus A (GBV-A), but not other flaviviruses, demonstrated an inhibitory effect on HCV infection. Through a series of sequence optimizations and modifications of the peptide helicity and hydrophobicity, we obtained a peptide designated GBVA10-9 with highly potent anti-HCV activity. GBVA10-9 suppressed infection with both cell culture-derived and pseudotyped HCV in vitro, and the 50% cell culture inhibitory concentration ranged from 20 nM to 160 nM, depending on the genotypic origin of the envelope proteins. GBVA10-9 had no detectable effects on either HCV attachment to Huh7.5.1 cells or viral RNA replication. No virucidal activity was found with GBVA10-9, suggesting an action mechanism distinct from that of C5A. The inhibitory effect of GBVA10-9 appeared to occur at the postbinding step during viral entry. Taken together, the results with GBVA10-9 demonstrated a potent activity for blocking HCV entry that might be used in combination with other antivirals directly targeting virus-encoded enzymes. Furthermore, GBVA10-9 also provides a novel tool to dissect the detailed mechanisms of HCV entry.

Recombinant human interleukin 28B: anti-HCV potency, receptor usage and restricted cell-type responsiveness

OBJECTIVES Interleukin 28B (IL28B) genetic variation has been recently reported as a potent predictor of hepatitis C virus (HCV) response to interferon (IFN) therapy. The aim of this study was to produce recombinant human IL28B (rhIL28B) in yeast and explore the action mechanisms of rhIL28B as a novel anti-HCV agent. METHODS A simple and efficient protocol for producing rhIL28B in the methylotrophic yeast Pichia pastoris was developed. The anti-HCV activity, induction of IFN-stimulated genes (ISGs), receptor usage and cellular responsiveness of rhIL28B were characterized. RESULTS The yield of secreted rhIL28B was optimized to 200 mg/L, and soluble rhIL28B that was approximately 95% pure was achieved using a one-step ion-exchange purification procedure. rhIL28B inhibited HCV propagation in Huh7.5.1 cells with an IC(50) of 0.15 × 10(-3) mg/L. Treatment of hepatoma cells with rhIL28B resulted in the phosphorylation of STAT1 within 1 h and expression of ISGs. The HCV inhibitory effects of rhIL28B were antagonized by the antibody neutralization of receptors IL10R2 and IL28R1. The combination of rhIL28B and ribavirin synergistically inhibited HCV production in cell culture. Importantly, compared with the broad-spectrum activity of IFN-α, we demonstrated restricted cell-type responsiveness of rhIL28B in liver, lung and prostate cells. CONCLUSIONS This study established an easy and highly efficient approach for the production of rhIL28B with potent in vitro antiviral activity and restricted cell tropism, and thus provides a novel antiviral candidate for improving the treatment of HCV-infected patients.

A novel small-molecule inhibitor of hepatitis C virus replication acts by suppressing signal transducer and activator of transcription 3

OBJECTIVES Hepatitis C virus (HCV) infects hepatocytes and causes liver damage. The aim of this study was to identify new classes of host-targeting anti-HCV compounds that may provide novel approaches for antiviral treatment regimens. METHODS Cell culture-derived HCV (HCVcc), replicons and pseudoparticles were used in combination with high-throughput screening, reporter gene assays and cytotoxicity and signalling pathway analyses. RESULTS A small-molecule inhibitor of HCV, N-(cyclopropyl(phenyl)methyl)thieno[2,3-d]pyrimidin-4-amine, designated IB-32, was identified by screening a compound library with a Jc1-luc HCVcc assay. By using various virus models, HCV replication was identified as the predominant step of IB-32s action. IB-32 inhibited HCVcc (genotype 2a) and HCV replicons (genotype 1b) at low nanomolar ranges (with IC50s of 40 ± 8 and 100 ± 15 nM, respectively). IB-32 was found to be non-toxic when tested against a panel of human cell lines in vitro at the effective antiviral dose. Mechanistically, IB-32 strongly inhibited STAT3 (Tyr705) phosphorylation, a necessary cellular factor for HCV replication and a pivotal therapeutic target for multiple cancers. Furthermore, the inhibition of HCV replication by IB-32 was augmented in cells with STAT3 knockdown. In contrast, the inhibitory effect of IB-32 was attenuated in cells overexpressing a constitutively active form of STAT3. CONCLUSION The results presented here identify a promising STAT3-targeting anti-HCV therapeutic candidate. This novel small molecule could be further optimized and developed for use as both an antiviral and an anti-cancer drug.

Albumin Fusion of Interleukin-28B: Production and Characterization of Its Biological Activities and Protein Stability

The cytokine interleukin-28B (IL-28B) has potential antiviral properties and regulatory roles in adaptive cellular immunity. A genome-wide association study identified a single nucleotide polymorphism near the IL-28B gene that strongly predicts response to hepatitis C treatment with interferon and ribavirin. In this study, we produced human serum albumin (HSA) fused to interleukin-28B (HSA-IL28B) in an attempt to determine the effects of albumin fusion on anti-Hepatitis C virus (HCV) activity and protein stability. HSA-IL28B was expressed at high levels in the yeast expression system we used and was easily purified. The biological activities of IL-28B were only retained when HSA was fused at the N-terminus. Compared with the native IL-28B, HSA-IL28B showed improved protein stability. HSA-IL28B inhibited HCV infection through the membrane receptors IL28R1and IL10R2. Additionally, we demonstrated that HSA-IL28B was able to induce interferon-stimulated genes, phosphorylate intracellular STAT1, and act in restricted cell types. Our findings highlight the potential clinical applications of the fusion protein during virus infection and for immune regulation.

Entry Properties and Entry Inhibitors of a Human H7N9 Influenza Virus

The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC50 of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.