Interactome map reveals phospholipid scramblase 1 as a novel regulator of hepatitis B virus x protein.

HBV X protein plays crucial roles during viral infection and hepatocellular carcinoma (HCC) development through interaction with various host factors. Here, we mapped the interactome of HBx using a yeast two-hybrid screen. Nine human proteins were identified as novel interacting partners of HBx, one of which is phospholipid scramblase 1 (PLSCR1). PLSCR1 is an interferon-inducible protein that mediates antiviral activity against DNA and RNA viruses. However, the molecular mechanisms of PLSCR1 activity against HBV remain unclear. Here, we reported that PLSCR1 promotes HBx degradation by a proteasome- and ubiquitin-dependent mechanism. Furthermore, we found that PLSCR1 inhibits HBx-mediated cell proliferation. After HBV infection, the protein level of PLSCR1 in plasma is elevated, and chronic hepatitis B patients with low plasma levels of PLSCR1 have a high risk of developing HCC. These results suggest that the nuclear trafficking of PLSCR1 mediates the antiviral activity and anticarcinogenesis against HBV by regulating HBx stability.

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.

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.

Detection of antibodies against porcine epidemic diarrhea virus in serum and colostrum by indirect ELISA.

An indirect porcine epidemic diarrhea virus (PEDV) anti-immunoglobulin (Ig) G ELISA based on the S1 portion of the spike protein was validated and compared with an indirect immunofluorescence assay. In serum samples from experimentally infected pigs (n = 35), anti-IgG PEDV antibodies were detected as early as 7 days post-infection. In field serum samples (n = 239), the diagnostic sensitivity of the S1 ELISA was 100% and the diagnostic specificity was 94%. The S1 ELISA showed no cross-reactivity with antibodies against other porcine coronaviruses. Colostrum samples (n = 133) were also tested for anti-PEDV IgG and IgA. The diagnostic sensitivity was 92% for IgG and 100% for IgA, and the diagnostic specificity was 90% for IgG and 99.4% for IgA. These data suggest that the S1 ELISA is a sensitive and specific test that could also be used to evaluate PEDV colostral immunity.

Study of the interaction between 8-azaguanine and bovine serum albumin using optical spectroscopy and molecular modeling methods.

The interaction between 8-azaguanine (8-Azan) and bovine serum albumin (BSA) in Tris-HCl buffer solutions at pH 7.4 was investigated by means of fluorescence and ultraviolet-visible (UV-Vis) spectroscopy. At 298 K and 310 K, at a wavelength of excitation (λ (ex)) of 282 nm, the fluorescence intensity decreased significantly with increasing concentrations of 8-Azan. Fluorescence static quenching was observed for BSA, which was attributed to the formation of a complex between 8-Azan and BSA during the binding reaction. This was illuminated further by the UV-Vis absorption spectra and the decomposition of the fluorescence spectra. The thermodynamic parameters ∆G, ∆H, ∆S were calculated. The results showed that the forces acting between 8-Azan and BSA were typical hydrophobic forces, and that the interaction process was spontaneous. The interaction distance r between 8-Azan and BSA, evaluated according to fluorescence resonance energy transfer theory, suggested that there is a high possibility of energy transfer from BSA to 8-Azan. Theoretical investigations based on homology modeling and molecular docking suggested that binding between 8-Azan and BSA is dominated by hydrophilic forces and hydrogen bonding. The theoretical investigations provided a good structural basis to explain the phenomenon of fluorescence quenching between 8-Azan and BSA.

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-3ß and Raf. Several HCC-related molecules, such as E-cadherin, ß-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.

Hepatitis C virus (HCV) infects human hepatocytes through several host factors. However, other prerequisite factors for viral entry remain to be identified. Using a yeast two-hybrid screen, we found that human phospholipid scramblase 1 interacts with HCV envelope proteins E1 and E2. These physical interactions were confirmed by co-immunoprecipitation and GST pull-down assays. Knocking down the expression of PLSCR1 inhibited the entry of HCV pseudoparticles. Moreover, PLSCR1 was required for the initial attachment of HCV onto hepatoma cells, where it specifically interacted with entry factor OCLN. We show that PLSCR1 is a novel attachment factor for HCV entry.