FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53

Abstract

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC).Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis. IMPORTANCEIt is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with the CHC background suggests that FBP1 has an important role in promoting HCV infection and HCC tumors by suppressing p53. H epatitis C virus (HCV) infection is a leading cause of chronic liver diseases. More than a decade after the identification of HCV as the major causative agent of non-A, non-B hepatitis (1), molecular strategies for complete eradication of HCV infection are actively pursued. HCV is the major cause of chronic liver disease. According to new findings from the U.S. Centers for Disease Control and Prevention (CDC), the number of individuals in the U.S. living with chronic hepatitis C virus infection is about 2.7 million (2). Globa...

Modulation of HCV replication and translation by ErbB3 binding protein1 isoforms

“…Nucleophosmin (NPM/B23) a multifunctional oncoprotein that is involved in regulating cell growth and proliferation (Okuwaki et al, 2002; Olanich et al, 2011), also functions as a positive and negative regulator of p53 and HDM2/MDM2 (Kurki et al, 2004). Earlier we have identified another cell factor, FUSE-binding protein, which represses NPM (Olanich et al, 2011) physically interacts with and antagonizes p53 function, and promotes persistent HCV replication (Dixit et al, 2015). The Ebp1-p48 isoform also negatively regulates p53 and thus may support HCV replication by antagonizing p53 function (Kim et al, 2010).…”

“…We used two sets of HepG2 cells (10 6 cells) cotransfected with plasmids expressing human CD81 (pTRIP-GFP-hCD81) and miR-122 (pTRIP-Puro-miR122). After 24 h post transfection, cells were layered with HCV-JFH1 virions in the presence of 5-µg/ml polybrene (Dixit et al, 2015). After 6 h, cells were washed two times with PBS, supplemented with fresh medium.…”

“…Ebp1-kd HepG2 cells were cotransfected with plasmids expressing human CD81 (pTRIP-GFP-hCD81) and miR-122 (pTRIP-Puro-miR122) with or without shRNA resistant expression clone of Ebp1-isoform. At 24 h posttransfection, cells were infected with HCV-JFH1 virions (Dixit et al, 2015). At 72h, total RNA was isolated to determine the level of HCV RNA, which was quantitated by q-RT-PCR; cell lysates were Western blotted for Ebp1 and actin.…”

“…Cells grown on 8-chamber tissue-culture slides for 24 h were fixed, treated with the primary anti-Ebp1 antibody (rabbit), then treated with anti-rabbit Alexa-594-labeled secondary antibody (red). The Ebp1-antibody treated cells were then separately treated with mouse anti-NS5A antibody (A), goat anti-NS5B (B), or mouse anti-PKR antibody (C), then treated with their respective Alexa 488-labeled secondary antibodies (Dixit et al, 2015). DAPI was used to stain nuclei.…”

Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection

“…Huh7 cells were either mock infected or infected with Jc1 and then transfected with either negative-control or TRIB3-specific siRNA. Thirty-six hours after siRNA transfection, cells were serum starved, and a wound-healing assay was then performed by scratching across the cell monolayer, as previously reported (22). As shown in Fig.…”