Hepatitis C virus (HCV) causes liver diseases, such as hepatitis, liver cirrhosis, steatosis, and hepatocellular carcinoma. To understand the life cycle and pathogenesis of HCV, the one-step growth of HCV in a cell culture system was analyzed using a highly infectious variant of the JFH1 clone. The observed profiles of HCV RNA replication indicated that the synthesis of negative-strand RNAs occurred at 6 h (h) after infection, followed by the active synthesis of positive-strand RNAs. Our measurements of infectious virus production showed that the latent period of HCV was about 12 h. The specific infectivity of HCV particles (focus-forming unit per viral RNA molecule) secreted to the extracellular milieu early in infection was about 30-fold higher than that secreted later during infection. The buoyant densities of the infectious virion particles differed with the duration of infection, indicating changes in the compositions of the virion particles.

High-mobility group box 1 (HMGB1), an abundant nuclear protein that triggers host immune responses, is an endogenous danger signal involved in the pathogenesis of various infectious agents. However, its role in hepatitis C virus (HCV) infection is not known. Here, we show that HMGB1 protein is translocated from the nucleus to cytoplasm and subsequently is released into the extracellular milieu by HCV infection. Secreted HMGB1 triggers antiviral responses and blocks HCV infection, a mechanism that may limit HCV propagation in HCV patients. Secreted HMGB1 also may have a role in liver cirrhosis, which is a common comorbidity in HCV patients. Further investigations into the roles of HMGB1 in the diseases caused by HCV infection will shed light on and potentially help prevent these serious and prevalent HCV-related diseases.Hepatitis C virus (HCV) is one of the major causative agents of hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) (17, 30). More than 170 million people are estimated to suffer from HCV infection worldwide (17). Chronic and persistent infection is a characteristic feature of HCV pathogenesis (30). During chronic infection, the production of virus particles is limited, and a restricted number of liver cells are infected. As a result, the viral dose in patients' blood generally is lower than that of other hepatitis-causing viruses, such as hepatitis B virus (HBV) (4). Moreover, a large portion of hepatocytes often remains uninfected by the virus even after long-term infection (28). These phenomena indicate the existence of balance between the HCV infection process and host mechanisms that protect against HCV infection. We speculate that innate and adaptive immunities contribute to the balance between infection and protection.High-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein that participates in DNA organization and the regulation of transcription. In addition to its nuclear function, HMGB1 plays an important role as a cytokine, mediating the responses to infection, injury, and inflammation (1, 2, 29, 42). HMGB1 is released passively from necrotic cells and is actively secreted from activated immune cells, such as macrophages, natural killer cells, and mature dendritic cells (2). The functionality of actively secreted HMGB1 is known to be modulated by posttranslational modifications, such as oxidation (2, 36). Extracellular HMGB1 can function by itself and/or in association with other molecules, including CpG DNA, lipopolysaccharide (LPS), and interleukin-1 (IL-1) (5). HMGB1 induces a variety of cellular responses that contribute to innate immunity, tissue repair, and cell migration through interactions with various receptors that activate multiple signal transduction responses. The Toll-like receptors (e.g., TLR2, TLR4, and TLR9) and the receptor for advanced glycation end products (RAGE) are known receptors for the cytokine functions of HMGB1 (2). TLR4, the major component of the LPS recognition receptor complex, engages in downstream signaling through My...

An infectious hepatitis C virus (HCV) cDNA clone (JFH1) was generated recently. However, quantitative analysis of HCV infection and observation of infected cells have proved to be difficult because the yield of HCV in cell cultures is fairly low. We generated infectious HCV clones containing the convenient reporters green fluorescent protein (GFP) and Renilla luciferase in the NS5a-coding sequence. The new viruses responded to antiviral agents in a dose-dependent manner. Responses of individual cells containing HCV to alpha interferon (IFN-␣) were monitored using GFP-tagged HCV and time-lapse confocal microscopy. Marked variations in the response to IFN-␣ were observed among HCV-containing cells.

We appended pyrene units covalently onto adenosine (forming A(P) units) and then incorporated them into oligonucleotides such that they were positioned in complementary locations in opposite strands in the middle positions of hairpin stems. System 1 (A(P)A(P)) behaves as an effective molecular beacon (MB) that changes color from green to blue upon duplex formation. In addition, we attached a cholesterol unit to a free terminus of one of these hairpins; this approach enhanced the cellular delivery of the modified MB relative to those encountered when using conventional transfection methods. These structurally simple cholesterol-based MB systems, which can be synthesized very efficiently, have good potential for opening up new and exciting opportunities in the field of in vivo biosensors.

Diabetic nephropathy, the major cause of chronic kidney disease, is associated with progressive renal fibrosis. Recently, accumulation of periostin, an extracellular matrix protein, was shown to augment renal fibrosis. Aptamers have higher binding affinities without developing the common side effects of antibodies. Thus, we evaluated the effect of periostin inhibition by an aptamer-based inhibitor on renal fibrosis under diabetic conditions. In vitro, transforming growth factor-β1 (TGF-β1) treatment significantly upregulated periostin, fibronectin, and type I collagen mRNA and protein expressions in inner medullary collecting duct (IMCD) cells. These increases were attenuated significantly in periostin-binding DNA aptamer (PA)-treated IMCD cells exposed to TGF-β1. In vivo, PA treatment attenuated the increased blood urea nitrogen levels in the diabetic mice significantly. Fibronectin and type I collagen mRNA and protein expressions increased significantly in the kidneys of diabetic mice: PA administration abrogated these increases significantly. Immunohistochemistry and Sirius Red staining also revealed that fibronectin expression was significantly higher and tubulointersititial fibrosis was significantly worse in diabetic mice kidneys compared with control mice. These changes were ameliorated by PA treatment. These findings suggested that inhibition of periostin using a DNA aptamer could be a potential therapeutic strategy against renal fibrosis in diabetic nephropathy.