Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes.To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3␤-hydroxysterol ⌬24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus. Hepatitis C virus (HCV)5 is composed of a single-stranded RNA genome of positive polarity (1). Translation of viral proteins is initiated from an internal ribosome entry site (2) and results in a single polypeptide that is subsequently cleaved by host and viral proteases to yield viable proteins (3). The HCV genome does not rely on canonical translation factors and can readily establish chronic infection without integrating into the host genome, resulting in hepatic steatosis and hepatocellular carcinoma (HCC) (4). More than 170 million people worldwide are infected with HCV (5); chronic HCV infection and aging are the major risk factors for HCC (6 -8). Liver cancer is the fifth most common cause of cancer mortality worldwide (9). The frequent inactivation of p53 in human HCC suggests that the loss of p53-dependent apoptosis may promote hepatocarcinogenesis (10). Chronic HCV infection results in chronic liver inflammation and induces endoplasmic reticulum stress and oxidative stress, which are thought to induce hepatocarcinogenesis (11, 12). The mechanistic details underlying HCC development are not fully understood. To gain insight into the molecular mechanisms underlying HCV-induced pathogenesis, we previously established RzM6 cells (13), a human hepatoblastoma (HepG2)-derived cell line in which expression of the full-length HCV genome is controlled by a Cre/loxP system. Expression of the HCV genome promoted anchorage-independent growth of RzM6 cells after 44 days of culture from the onset of HCV expression (RzM6-44d cells) but not in RzM6 cells after 0 days (RzM6-0d cells) (13). In the present study, we generated monoclonal antibodies against RzM6 cells cultured for longer than 44 days (RzM6-LC cells) and then screened the antibodies for their ability to...

Extrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%- IntroductionMore than 175 million people worldwide are infected with hepatitis C virus (HCV), a positive-strand RNA virus that infects both hepatocytes and peripheral blood mononuclear cells. 1 Chronic HCV infection may lead to hepatitis, liver cirrhosis, hepatocellular carcinomas 2,3 and lymphoproliferative diseases such as B-cell non-Hodgkin lymphoma and mixed-cryoglobulinemia. 1,4-6 B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection 7 with geographic and ethnic variability. 8,9 Based on a meta-analysis, the prevalence of HCV infection in patients with B-cell non-Hodgkin lymphoma is approximately 15%. 8 The HCV envelope protein E2 binds human CD81, 10 a tetraspanin expressed on various cell types including lymphocytes, and activates B-cell proliferation 11 ; however, the precise mechanism of disease onset remains unclear. We previously developed a transgenic mouse model that conditionally expresses HCV cDNA (nucleotides 294-3435), including the viral genes that encode the core, E1, E2, and NS2 proteins, using the Cre/loxP system (in coreϳNS2 [CN2] mice). 12,13 The conditional transgene activation of the HCV cDNA (core, E1, E2, and NS2) protects mice from Fas-mediated lethal acute liver failure by inhibiting cytochrome c release from mitochondria. 13 In HCV-infected mice, persistent HCV protein expression is established by targeted disruption of irf-1, and high incidences of lymphoproliferative disorders are found in CN2 irf-1 ؊/؊ mice. 14 Infection and replication of HCV also occur in B cells, 15,16 although the direct effects, particularly in vivo, of HCV infection on B cells have not been clarified.To define the direct effect of HCV infection on B cells in vivo, we crossed transgenic mice with an integrated full-length HCV genome (Rz) under the conditional Cre/loxP expression system with mice expressing the Cre enzyme under transcriptional control of the B lineage-restricted gene CD19, 17 we addressed the effects of HCV transgene expression in this study. Methods Animal experimentsWild-type (WT), Rz, CD19Cre, RzCD19Cre mice (129/sv, BALB/c, and C57BL/6J mixed background), and MxCre/CN2-29 mice (C57BL/6J background) were maintained in conventional animal housing under specific pathogen-free conditions. All animal experiments were performed according to the guidelines of the Tokyo Metropolitan Institute of Medical Science or the Kumamoto University Subcommittee for Laboratory Animal Care. The protocol was approved by the Institutional Review Boards of both facilities. Measurements of HCV protein and RNAMice were anesthetized and bled, and tissues (spleen, lymph nodes, liver, and tumors) were homogenized in lysis buffer (1% sodium dodecyl sulfate; 0.5% (wt/vol) nonyl phenoxypolyethoxylethanol; 0.15M NaCl; 10 mM For personal use only. on May 9, 2018. by guest www.bloodjournal.org From tris(hydroxymethyl)aminomethane, pH 7.4) using a Dounce homogenizer. The concentration of HCV ...

BACKGROUND & AIMS The molecular mechanisms of lymphoproliferation associated with the disruption of interferon (IFN) signaling and chronic hepatitis C virus (HCV) infection are poorly understood. Lymphomas are extrahepatic manifestations of HCV infection; we sought to clarify the molecular mechanisms of these processes. METHODS We established interferon regulatory factor-1– null (irf-1−/−) mice with inducible and persistent expression of HCV structural proteins (irf-1/CN2 mice). All the mice (n = 900) were observed for at least 600 days after Cre/loxP switching. Histologic analyses, as well as analyses of lymphoproliferation, sensitivity to Fas-induced apoptosis, colony formation, and cytokine production, were performed. Proteins associated with these processes were also assessed. RESULTS Irf-1/CN2 mice had extremely high incidences of lymphomas and lymphoproliferative disorders and displayed increased mortality. Disruption of irf-1 reduced the sensitivity to Fas-induced apoptosis and decreased the levels of caspases-3/7 and caspase-9 messenger RNA species and enzymatic activities. Furthermore, the irf-1/CN2 mice showed decreased activation of caspases-3/7 and caspase-9 and increased levels of interleukin (IL)-2, IL-10, and Bcl-2, as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes. IL-2 and IL-10 were induced by the HCV core protein in splenocytes. CONCLUSIONS Disruption of IFN signaling resulted in development of lymphoma, indicating that differential signaling occurs in lymphocytes compared with liver. This mouse model, in which HCV expression and disruption of IFN signaling synergize to promote lymphoproliferation, will be an important tool for the development of therapeutic agents that target the lymphoproliferative pathway.

Hepatitis C virus (HCV) infection leads to the development of hepatic diseases, as well as extrahepatic disorders such as B-cell non-Hodgkin's lymphoma (B-NHL). To reveal the molecular signalling pathways responsible for HCV-associated B-NHL development, we utilised transgenic (Tg) mice that express the full-length HCV genome specifically in B cells and develop non-Hodgkin type B-cell lymphomas (BCLs). The gene expression profiles in B cells from BCL-developing HCV-Tg mice, from BCL-non-developing HCV-Tg mice, and from BCL-non-developing HCV-negative mice were analysed by genome-wide microarray. In BCLs from HCV-Tg mice, the expression of various genes was modified, and for some genes, expression was influenced by the gender of the animals. Markedly modified genes such as Fos, C3, LTβR, A20, NF-κB and miR-26b in BCLs were further characterised using specific assays. We propose that activation of both canonical and alternative NF-κB signalling pathways and down-regulation of miR-26b contribute to the development of HCV-associated B-NHL.

Our results suggest that 2-152a MAb suppresses HCV replication and infection through BGT-1. These findings highlight important roles of BGT-1 in HCV replication and reveal a possible target for anti-HCV therapy.