Hepatitis B Virus Hijacks Cellular Factors And Signal Pathways To Evade Host Immunity And Maintain Persistent Infection

Hepatitis B virus (HBV) infection is one of the major causes of liver disease, ranging from fulminant hepatitis to cirrhosis, and eventually hepatocellular carcinoma (HCC). Here we study the relationship between HBV and collagen triple helix repeat containing 1 (CTHRC1) gene. CTHRC1 was a secreted protein with multiple functions, and also strongly associated with invasion and metastasis in human cancers. However, the effect of CTHRC1 on regulating virus replication has not been investigated and reported. Our work aims at the clarity of the regulation mechanism of HBV-induced CTHRC1 expression and in turn CTHRC1-affected HBV replication.The effect of HBV infection on CTHRC1 expression was investigated and confirmed. First, CTHRC1 protein levels were found higher in the sera of patients with chronic hepatitis B (CHB) (n=153) than in healthy individuals (n=164). Second, CTHRC1 mRNA levels were elevated in liver biopsy specimens from HBV-associated HCC tissues (n=12) than in noncancerous tissues (n=13). Third, CTHRC1 protein levels were positively correlated with HBV DNA loads in the sera of 142 HBV-infected patients. In addition, CTHRC1 protein levels were calcuated in HBV-transfected HepG2 cells and HepG2.2.15 cells carrying HBV genome. Thus, CTHRC1 expression was activated in patients infected with HBV and in hepatoma cells carrying HBV genome.The molecular mechanism by which HBV activates CTHRC1 expression was elucidated. CTHRC1 promoter activity, mRNA expression and protein production were up-regulated by DNMT inhibitor,5’-aza-dc, indicating that DNMT plays a role in CTHRC1 activation. Genomic DNA sequencing analysis indicated that the percentage of methylation of CTHRC1 promotor was lower in HCC tissues than in noncancerous tissues. Furthermore CTHRC1 promoter activity, mRNA expression and protein production were strongly repressed by DNMT3a, but activated by siDNMT3a, confirming that DNMT3a is required for CTHRC1 expression.Then we first time explored the effect of CTHRC1 on viral replication. HBV e antigen (HBeAg), HBV s antigen (HBsAg) and HBV core-associated DNA were up-regulated by CTHRC1 in HepG2, HepG2.215 and L02 cells in dose- and time-dependent fashions, but down-regulated by siCTHRC1. HBV specific transcripts of 4.1/3.5 and 2.4/2.1kb were predominantly enhanced by CTHRC1, but reduced by siCTHRC1. Thus, over-expression of CTHRC1 resulted in activating HBV replication and knock-down of CTHRC1 led to suppressing HBV replication.To reveal the molecular mechanism involved in CTHRC1-mediated HBV replication, we evaluated the effects of signaling components on such regulation. Results showed that HBeAg and HBsAg were activated by CTHRC1, repressed by U0126 (MEK1/2 inhibitor), SP600125 (JNK inhibitor) and GF109203 (PKC inhibitor), suggesting that PKC、ERK and JNK are involved in CTHRC1-activated HBV replication. Further we revealed that HBeAg and HBsAg were up-regulated by CTHRC1, but repressed by si-c-Jun; and that antibody against p-c-Jun was immunoprecipitated with AP-1 binding sequence of HBV genome. Taken together, we demonstrated that PK、ERK、JNK and c-Jun are involved in CTHRC1-activated HBV replication.It is known that HBV replication is strongly inhibited in response to interferon. Thus, we investigated the role of CTHRC1 in regulating the anti-HBV activity of IFN. We showed that HBeAg and HBsAg expression and HBV core-associated DNA were down-regulated by IFN-a (type I IFN), IFN-y (type II IFN) and IFN-λ1 (type III IFN), respectively, but their inhibitory effects were reduced by CTHRC1. Further results indicated that the expression of IFN-α/β receptors (IFNARa/p) and IFN-stimulated proteins (PKR, Mx1 and OAS1) and the phosphorylation of STAT1/2 were up-regulated by IFN-a, but down-regulated by CTHRC1. In addition, both phosphorylation and ubiquitination of INFARawere enhanced by CTHRC1, but repressed by GF109203 and U0126. Taken together, we revealed that CTHRC1 facilitates HBV replication by activating the PKC/ERK/JNK/c-Jun signaling cascade to suppress IFN/JAK/STAT antiviral activity.In summary, HBV activates CTHRC1 expression by epigenetic modification and transcription regulation. CTHRC1 in turn facilitates HBV replication by stimulating PKC/ERK/JNK/c-Jun cascade, attenuating IFN/JAK/STAT activity, and enhancing IFN-α/β degradation. Thus, we revealed a novel mechanism by which HBV hijacks cellular factors and signal cascades to evade host antiviral immunity and maintain persistent infection. In addition, we discovered a new role of CTHRC1 in HBV infection in the liver cells.