Dynamics of HBV cccDNA expression and transcription in different cell growth phase

<p>Abstract</p> <p>Background</p> <p>The covalently closed-circular DNA (cccDNA) of hepatitis B virus (HBV) is associated with viral persistence in HBV-infected hepatocytes. However, the regulation of cccDNA and its transcription in the host cells at different growth stages is not well understood.</p> <p>Methods</p> <p>We took advantages of a stably HBV-producing cell line, 1.3ES2, and examine the dynamic changes of HBV cccDNA, viral transcripts, and viral replication intermediates in different cellular growth stages.</p> <p>Results</p> <p>In this study, we showed that cccDNA increased suddenly in the initial proliferation phase of cell growth, probably attributable to its nuclear replenishment by intracellular nucleocapsids. The amount of cccDNA then decreased dramatically in the cells during their exponential proliferation similar to the loss of extrachromosomal plasmid DNA during cell division, after which it accumulated gradually while the host cells grew to confluency. We found that cccDNA was reduced in dividing cells and could be removed when proliferating cells were subjected to long term of lamivudine (3TC) treatment. The amounts of viral replicative intermediates were rapidly reduced in these proliferating cells and were significantly increased after cells reaching confluency. The expression levels of viral transcripts were increased in parallel with the elevated expression of hepatic transcription factors (HNF4α, CEBPα, PPARα, etc.) during cell growth confluency. The HBV transcripts were transcribed from both integrated viral genome and cccDNA, however the transcriptional abilities of cccDNA was less efficient then that from integrated viral genome in all cell growth stages. We also noted increases in the accumulation of intracellular viral particles and the secretion of mature virions as the cells reached confluency and ceased to grow.</p> <p>Conclusions</p> <p>Based on the dynamics of HBV replication, we propose that HBV replication is modulated differently in the different stages of cell growth, and can be divided into three phases (initial proliferation phase, exponential proliferation phase and growth confluency phase) according to the cell growth curve. The regulation of cccDNA in different cell growth phase and its importance regarding HBV replication are discussed.</p

Pretreatment with a Heat-Killed Probiotic Modulates the NLRP3 Inflammasome and Attenuates Colitis-Associated Colorectal Cancer in Mice.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Inflammation contributes to cancer development and inflammatory bowel disease is an important risk factor for CRC. The aim of this study is to assess whether a widely used probiotic Enterococcus faecalis can modulate the NLRP3 inflammasome and protect against colitis and colitis-associated CRC. We studied the effect of heat-killed cells of E. faecalis on NLRP3 inflammasome activation in THP-1-derived macrophages. Pretreatment of E. faecalis or NLRP3 siRNA can inhibit NLRP3 inflammasome activation in macrophages in response to fecal content or commensal microbes, P. mirabilis or E. coli, according to the reduction of caspase-1 activation and IL-1β maturation. Mechanistically, E. faecalis attenuates the phagocytosis that is required for the full activation of the NLRP3 inflammasome. In in vivo mouse experiments, E. faecalis can ameliorate the severity of intestinal inflammation and thereby protect mice from dextran sodium sulfate (DSS)-induced colitis and the formation of CRC in wild type mice. On the other hand, E. faecalis cannot prevent DSS-induced colitis in NLRP3 knockout mice. Our findings indicate that application of the inactivated probiotic, E. faecalis, may be a useful and safe strategy for attenuation of NLRP3-mediated colitis and inflammation-associated colon carcinogenesis

Transforming Growth Factor-β1 Suppresses Hepatitis B Virus Replication by the Reduction of Hepatocyte Nuclear Factor-4α Expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene

The HNF-4α binding sites within HBV core promoter are the essential elements for the inhibitory effects of TGF-β1.

<p>For the promoter activity assay, HepG2 cells were transfected with luciferase reporter plasmids driven by HBV core promoter (CP) and enhancer I/X promoter (EnI/X) (A), with luciferase reporter plasmids driven by HBV core promoter (CP) and truncated HBV core promoters (CPD1, CPD2 and CPD3) (B), or with luciferase reporters with HNF4BE(s)-mutated HBV core promoters (NEm, Npm and NEpm) (C). Two days after TGF-β1 treatment, the cell lysate was extracted for luciferase activity analysis. The luciferase activities from cells transfected with different reporter plasmids were normalized to the galactosidase activities from co-transfected pCMV-beta-galactosidase plasmids, and the relative luciferase activities were compared to that of core promoter (CP) or enhancer I/X promoter (EnI/X), respectively. The relative ratio of their promoter activities between TGF-β1-treated cells and corresponding mock control cells were shown below. The schematic illustration shows the reporter constructs with deletions or specific mutations of HNF-4α binding sites in the HBV core promoter region. The results were shown as relative ratio of the firefly luciferase activities normalized to the beta-galactosidase activities in triplicates (mean value±S.D). Statistical analyses were carried out by 2-sided paired t test, and the star symbol representing the <i>p</i> values less than 0.01 were considered as statistically significant.</p

TGF-β1 treatment represses HNF-4α expression in HBV-producing cells.

<p>(A) The binding activity of endogenous HNF-4α in the presence or absence of TGF-β1 was examined by EMSA analysis. Nuclear extracts were prepared from HepG2 cells with or without TGF-β1 treatment for 2 days, and then incubated with HBV-specific probes containing HNF-4α binding element. Lane 1 is a free probe control without any nuclear extracts. Lane 2 is the specific probe incubated with HepG2 nuclear extracts. Lane 3 is the specific probe incubated with TGF-β1-treated nuclear extracts. Lane 4 is the specific probe incubated with HepG2 nuclear extracts and antibody against HNF-4α. Lane 5 is the specific probe incubated with HepG2 nuclear extracts and 100-fold molar excess cold competitor. (B) 1.3ES2 cells were plated followed by TGF-β1 treatment for 6 days. Total RNA was extracted analyzed by Northern blot with an HNF-4α specific probe. The expression of GAPDH was used as loading control. (C) 1.3ES2 cells were treated with TGF-β1 for 6 days. To examine the expression of HNF-4α, the cell lysate was extracted and subjected by Western blot analysis. The loaded amount of total protein was adjusted by the expression level of actin. (D) 1.3ES2 cells were infected with lentivirus carrying HNF-4α shRNA. The transduced cells were selected with puromycin, and then cells were treated by TGF-β1 for 6 days. The cell lysate was collected for Western blot analysis to determine the expression level of HNF-4α and HBc. The total protein loaded was adjusted by actin expression level.</p

HNF-4α plays a crucial role in the TGF-β1-associated inhibitory effect on HBV replication.

<p>1.3ES2 cells were treated with or without 10 µM MG132 for 1 hour and TGF-β1 for 12 hours. (A) The cell lysate was collected for Western blot analysis for the detection of HNF-4α and HBc. The loading amount of total protein was monitored by actin. (B) Total RNA was extracted, and the expression of HBV transcripts were revealed by Northern blot with using an HBV-specific probe. The expression of GAPDH was used as the loading control. (C) HepG2 cells were transfected with pHBV1.3 plasmids and an increased amount of rat HNF-4α-expressing plasmids. After treated with TGF-β1 for 2 days, the cell lysate was collected for the detection of HNF-4α and HBc by Western blot analysis. The expression of core particles was examined by particle blot analysis. The equal amount of total protein was revealed by the level of actin. The amount of HBc and HBV particle were also adjusted by the expression level of EGFP for monitoring the transfection efficiency. (D) Total RNA was extracted, and the expression of HBV transcripts were revealed by Northern blot with an HBV-specific probe. The expression of the kanamycin-neomycin phosphate transferase (kan/neo) was used as the loading control. Total DNA was extracted followed by HindIII digestion. The viral replicative intermediates were examined by Southern blot analysis using an HBV-specific probe. Bands corresponding to the relaxed circular double-stranded DNA (RC), the replicative intermediates are indicated individually.</p

The HNF-4α binding sites within the HBV core promoter are essential elements for the TGF-β1-mediated suppression.

<p>(A) Schematic illustration of HBV-expressing plasmids with HNF-4α binding sites mutations within the HBV core promoter region. (B) Stably HBV-producing cell lines (1.3ES2 and 1.3NEpm) were treated with or without TGF-β1 for 6 days. To determine the expression level of HBc, cell lysate was extracted and subjected to Western blot analysis. The loaded amount of total protein was adjusted by the expression level of actin. (C) Total DNA was extracted from stable HBV-producing cell lines followed by HindIII digestion, and the viral replicative intermediates were examined by Southern blot analysis using an HBV-specific probe. The integrated HBV genomes were revealed as the internal control for equal amount of sample loading. Bands corresponding to the integrated genome (integrated), the relaxed circular double-stranded DNA (RC), the replicative intermediates are indicated individually.</p