Tridimensional infiltration of DNA viruses into the host genome shows preferential contact with active chromatin

Whether DNA viruses contact specific regions of host genomes or make random contacts is unclear. Here, the authors use Hi-C and show that HBV cccDNA and Ad5 DNA contact preferentially active chromatin at CpG islands for the former and at transcription start sites and enhancers for the latter

ANTI CAPSID DRUGS HAP12 AND AT130 TARGET HBV CORE PROTEIN NUCLEAR FUNCTIONS

Introduction and aim: HBV core protein (Cp) represents an attractive new therapeutic target for HBV chronic infection. Cp has been shown to bind the nuclear cccDNA mini-chromosome as well as a number of cellular genes promoters. Several compounds that target Cp and HBV capsids assembly, including the hetero- aryl-dihydropyrimidines (HAPs) and the phenyl-propenamide derivatives AT61 and AT130, have been shown to inhibit HBV repli- cation in vitro and in vivo. HAPs and AT130 enhance the rate of Cp assembly and stabilize preferentially non-capsid polymers of Cp. Here we investigated the ability of the Core protein Assembly Mod- ulators (CaMPs) HAP12 and AT130 to affect both nuclear cccDNA transcription and cytoplasmic capsid assembly Cp functions. Methods: HAP12 and AT130 effects on capsid-associated HBV- DNA, cccDNA and pgRNA levels (quantitative real-time PCR with specific primers) were assessed in: (a) HBV-infected NTCP-HepG2 cells; (b) AD38 inducible HBV stable cell line. Recruitment of HBc and histone modifications on the viral minichromosome were assessed using the cccDNA ChIP assay in AD38 cells. Results: CaMPs treatments resulted in a very strong inhibition of HBV replication (>95%) and a significant but incomplete reduction of the stable cccDNA pool. A strong effect on cccDNA-dependent HBeAg production (AD38 tet-off) and pgRNA transcription (AD38 tet-off/tet-on and NTCP-HepG2 infected cells) was also demon- strated. The ability of HAP12 to target cccDNA transcription was confirmed by the reduced cccDNA-bound H3 histone acetylation and the decreased HBc occupancy on the cccDNA in induced AD38 cells. Importantly, when CaMPs treatment was started during infec- tion, cccDNA formation/accumulation was completely inhibited (>95%) and viral replication was blunted. Conclusions: Anti-capsid compounds (CpAMs) have an impact on Cp nuclear functions at multiple levels: block of new cccDNA formation/accumulation, reduction of an established cccDNA pool and inhibition of HBc occupancy and histone acetylation on the cccDNA that translate into a reduced pgRNA transcription

IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome

The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTβ) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes

A newly identified flower-specific splice variant of AUXIN RESPONSE FACTOR8 regulates stamen elongation and endothecium lignification in arabidopsis

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana. By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26. Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes

Modulation of P-STAT3 chromatin binding by IL6.

<p>In upper panel, <b>A)</b> Anti-STAT3 and anti-P-STAT3 chromatin immuno-precipitations were performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142599#pone.0142599.g002" target="_blank">Fig 2A</a> and analyzed with cccDNA specific primers (see Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142599#pone.0142599.g001" target="_blank">Fig 1</a>). <b>B)</b> HepG2 cells were transfected with monomeric linear full-length HBV DNA in combination with the indicated siRNA pools. After 48 hours, total RNA was extracted and pgRNA levels analyzed by qPCR as described in Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142599#pone.0142599.g001" target="_blank">Fig 1D</a>. <b>C)</b> Anti-STAT3 and anti-P-STAT3 immuno-precipitates were analyzed with primers specific for the Haptoglobin (HP) promoter. All ChIP results are expressed as Fold Induction (FI) of the % of Input and the histograms show the mean from three independent experiments; bars indicate S.D. <b>D)</b> 30 μg of nuclear proteins were analyzed by immunoblot with anti-STAT3, anti-P-STAT3 and anti-lamin B (loading control) antibodies (<i>left panel</i>). Densitometric quantitation of STAT3, P-STAT3 and lamin B immunoblots are shown in the <i>right panel</i>.</p

IL-6 inhibits cccDNA transcription activity in HBV infected NTCP-HepG2 cells.

<p>HepG2-NTCP cells were infected with 6 × 10² genome equivalents/cells of HBV in and treated with rIL6 for 48 hours at day 10 post-infection. <b>A)</b> Cytoplasmic HBV core particles were isolated from untreated and IL6 treated infected cells and total core particles associated HBV DNA was quantified as described in the Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142599#pone.0142599.g001" target="_blank">Fig 1C</a>). <b>B)</b> RNAs were isolated from untreated and IL6-treated HepG2-NTCP infected cells. The 3.5Kb/pgRNA and the pre-S/S RNA were quantified and described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142599#pone.0142599.g001" target="_blank">Fig 1D</a>. <b>C)</b> cccDNA was extracted from the nuclei of untreated and IL6-treated infected cells. qPCR analysis was performed using cccDNA selective primers and β-globin primers to normalize the DNA samples. All results in <b>A-C)</b> are expressed as arbitrary units and the histograms show the mean from three independent experiments; bars indicate S.D. <b>D)</b> Cross-linked chromatin is extracted from the nuclei of NTCP-HepG2 cells treated or not with with rIL6 for 48 hours at day 10 post-infection. The cross-linked chromatin was immunoprecipitated with a relevant control IgG or specific anti-AcH3 and anti-HDAC1 antibodies. ChIPs were analyzed and the results expressed as described in the Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142599#pone.0142599.g001" target="_blank">Fig 1F</a>. * 0,01 ≤ P < 0,05; ** 0,001 ≤ P < 0.01; *** P < 0,001.</p

Schematic model of IL6 modulation of cccDNA transcription.

<p>STAT3, HNF1α and HNF4α bind the cccDNA and contribute to activate the transcription of cccDNA (middle). IL6 treatment results in the hypo-acetylation of cccDNA-bound histones and inhibits HBV transcription through the combined effect on HNF1α and HNF4α protein levels and a lower recruitment of P-STAT3 to the cccDNA, as compared to cellular promoters (i.e. Haptoglobin).</p