The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Salpini R ，Surdo M ，Cortese MF ，Palumbo GA ，Carioti L ，Cappiello G ，Spanò A ，Trimoulet P ，Fleury H ，Vecchiet J ，Pasquazzi C ，Mirabelli C ，Scutari R ，Sacco A ，Alkhatib M ，Missale G ，Francioso S ，Sarmati L ，Andreoni M ，Angelico M ，Ceccherini-Silberstein F ，Levrero M ，Perno CF ，Belloni L ，Svicher V ... -  《-》

Hepatitis B virus X protein-elevated MSL2 modulates hepatitis B virus covalently closed circular DNA by inducing degradation of APOBEC3B to enhance hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) infection is a leading cause in the occurrence of hepatitis B, liver cirrhosis, and liver cancer, in which nuclear HBV covalently closed circular DNA (cccDNA), the genomic form that templates viral transcription and sustains viral persistence, plays crucial roles. In the present study, we explored the hypothesis that HBV X protein (HBx)-elevated male-specific lethal 2 (MSL2) activated HBV replication by modulating cccDNA in hepatoma cells, leading to hepatocarcinogenesis. Immunohistochemical analysis revealed that the expression of MSL2 was positively associated with that of HBV and was increased in the liver tissues of HBV-transgenic mice and clinical HCC patients. Interestingly, microarray profiling identified that MSL2 was associated with those genes responding to the virus. Mechanistically, MSL2 could maintain HBV cccDNA stability through degradation of APOBEC3B by ubiquitylation in hepatoma cells. Above all, HBx accounted for the up-regulation of MSL2 in stably HBx-transfected hepatoma cell lines and liver tissues of HBx-transgenic mice. Luciferase reporter gene assays revealed that the promoter region of MSL2 regulated by HBx was located at nucleotide -1317/-1167 containing FoxA1 binding element. Chromatin immunoprecipitation assay validated that HBx could enhance the binding property of FoxA1 to MSL2 promoter region. HBx up-regulated MSL2 by activating YAP/FoxA1 signaling. Functionally, silencing MSL2 was able to block the growth of hepatoma cells in vitro and in vivo. HBx-elevated MSL2 modulates HBV cccDNA in hepatoma cells to promote hepatocarcinogenesis, forming a positive feedback loop of HBx/MSL2/cccDNA/HBV. Our finding uncovers insights into the mechanism by which MSL2 as a promotion factor in host cells selectively activates extrachromosomal DNA. (Hepatology 2017;66:1413-1429).

Gao Y ，Feng J ，Yang G ，Zhang S ，Liu Y ，Bu Y ，Sun M ，Zhao M ，Chen F ，Zhang W ，Ye L ，Zhang X ... -  《-》

A novel mutant 10Ala/Arg together with mutant 144Ser/Arg of hepatitis B virus X protein involved in hepatitis B virus-related hepatocarcinogenesis in HepG2 cell lines.

Hepatitis B virus (HBV) infection-related hepatocellular carcinoma (HCC) represents a major health problem worldwide. HBV X (HBx) protein is the most common open reading frame that may undergo mutations, resulting in the development of HCC. This study aimed to determine specific HBx mutations that differentiate the central- and para-tumor tissues, and identify their association with HCC development. HBx gene from HCC tumor and para-tumor tissues of 47 HCC patients was amplified, sequenced and statistically analyzed. A novel combination of 2 mutations at residues 10 and 144 was identified which might play a significant role in HCC development. Expression vectors carrying HBx with the specific mutations were constructed and transfected into HepG2 and p53-null HepG2 cells. Compared to wild type (WT) and single mutation of HBx at residue 10 or 144, the 10/144 double mutations strongly up-regulated p21 expression and prolonged G1/S transition in WT- and p53-null HepG2 cells. Apoptosis was also inhibited by HBx harboring 10/44 double-mutation. Binding of 10/144 double-mutant HBx to p53 was lower than WT HBx. Conclusively, the 10/144 double mutation of HBx might play a crucial role in HCC formation.

Shi Y ，Wang J ，Wang Y ，Wang A ，Guo H ，Wei F ，Mehta SR ，Espitia S ，Smith DM ，Liu L ，Zhang Y ，Chen D ... -  《-》

Hepatitis B virus replicating in hepatocellular carcinoma encodes HBx variants with preserved ability to antagonize restriction by Smc5/6.

Hepatitis B virus infection is a major cause of liver diseases including hepatocellular carcinoma (HCC). The viral regulatory protein HBx is essential for viral replication and has been involved in the development of HCC. Recently, we characterized a subset of HCCs that replicate HBV. Our aim was to characterize HBx encoded by the full-length HBV DNA (cccDNA) in HCC and non-HCC liver. HBx genes were amplified and sequenced from eight paired HCC and non-HCC tissues in which HBV cccDNA and pgRNA were both present. Sequence analyses identified twelve amino acid positions mutated between HCC and non-HCC liver, and detected in at least three cases. We next assessed the impact of these mutations on HBx function by testing their transcriptional activity. We examined their ability to rescue the transcription of HBV virus deficient for HBx in differentiated HepaRG cells and to induce Smc5/6 degradation, which is mandatory for viral replication. We assessed their capacity to activate a CREB-dependent reporter. Finally we analyzed their growth suppressive activity using colony formation assays. Our results showed that most HBx variants isolated from HCC retain their ability to support HBV cccDNA transcription and to degrade Smc5/6. Strikingly, HCC specific HBx variants are impaired in their antiproliferative activity, which may be detrimental for tumor growth. In conclusion, in contrast to previous observations that tumor HBx variants lack transcriptional activity, we showed here that HBx variants have retained their ability to counteract Smc5/6 and thus to activate cccDNA transcription although they tend to lose antiproliferative activity.

Rivière L ，Quioc-Salomon B ，Fallot G ，Halgand B ，Féray C ，Buendia MA ，Neuveut C ... -  《-》

Distinct distribution pattern of hepatitis B virus genotype C and D in liver tissue and serum of dual genotype infected liver cirrhosis and hepatocellular carcinoma patients.

Datta S ，Roychoudhury S ，Ghosh A ，Dasgupta D ，Ghosh A ，Chakraborty BC ，Ray S ，Gupta S ，Santra AK ，Datta S ，Das K ，Dhali GK ，Chowdhury A ，Banerjee S ... -  《PLoS One》