The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.
Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.
Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti-HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV-positive plasma. This anti-HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti-HCV therapy. (HEPATOLOGY 2003;38:1282-1288 P ersistent infection with the hepatitis C virus (HCV), identified as the major causative agent of non-A, non-B hepatitis, 1,2 has been closely related to liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. 3 The development of these liver diseases from HCV carriers, an estimated 170 million people throughout the world, is a major public health problem. Effective anti-HCV therapy has been restricted mainly to therapy with interferon (IFN) and a combination of IFN and ribavirin. However, because the virus is not eliminated from approximately one half of HCVinfected patients treated with these agents, 4 alternative approaches to the treatment of HCV infection are needed.Recently, an HCV subgenomic replicon cell culture system has been established in which an HCV subgenomic replicon autonomously replicated in Huh-7 cells (HCV replicon cells). 5 This replicon is composed of the HCV 5Ј-untranslated region containing an internal ribosomal entry site, the neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural proteins (NS) 3 through NS5B; and the HCV 3Ј-untranslated region (Fig. 1A). This system provides a unique tool for studying the mechanisms of HCV replication and screening as well as evaluating anti-HCV compounds. Taking advantage of this feature, we examined the effects of various types of compounds on the replication of HCV using HCV replicon cells established in our laboratory 6 (Miyanari et al., manuscript accepted for publication). Consequently, we found that a well-known immunosuppressant, cyclosporin A (CsA), 7 had a strong suppressive effect on HCV replication in these cells. Moreover, we found suppressive activity of CsA for multiplication of the HCV genome in cultured human hepatocytes infected with HCV. The mechanism of the anti-HCV activity of CsA was also studied. Materials and MethodsCell Culture. Huh-7 and MH-14 cells, HCV replicon cells, were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum. PH5CH8 cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle medium and F12 medium supplemented with 100 ng/mL epidermal gro...
Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.
Chronic hepatitis B virus (HBV) infection is a global public health challenge on the same scale as tuberculosis, HIV, and malaria. The International Coalition to Eliminate HBV (ICE-HBV) is a coalition of experts dedicated to accelerating the discovery of a cure for chronic hepatitis B. Following extensive consultation with more than 50 scientists from across the globe, as well as key stakeholders including people affected by HBV, we have identified gaps in our current knowledge and new strategies and tools that are required to achieve HBV cure. We believe that research must focus on the discovery of interventional strategies that will permanently reduce the number of productively infected cells or permanently silence the covalently closed circular DNA in those cells, and that will stimulate HBV-specific host immune responses which mimic spontaneous resolution of HBV infection. There is also a pressing need for the establishment of repositories of standardised HBV reagents and protocols that can be accessed by all HBV researchers throughout the world. The HBV cure research agenda outlined in this position paper will contribute markedly to the goal of eliminating HBV infection worldwide.
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