Regulation of viral expression by the HBV core protein and the characterization HBc as a potential therapeutic target for HBV cure

Background and Aim The hepatitis B virus (HBV) is a DNA virus organized into nucleosomal structures. HBV produces covalently closed double-stranded DNA (cccDNA) that is found in the nuclei of infected cells as a viral minichromosome. HBV encoded proteins HBx and HBc both bind the cccDNA early after infection. HBx is required for the inactivation of the HBV cellular restriction factor smc5/6 and to establish and maintain cccDNA transcription. HBc binding to the cccDNA in thought to contribute to cccDNA chromatinization and nucleosome spacing whereas its impact on cccDNA transcription is still debated. Increasing evidence indicate that HBV proteins regulating viral minichromosomes also interact with the cellular chromatin and chromatin modifying enzymes to target cellular genes expression through epigenetic modifications. A ChIP-Seq genome wide analysis of HBx chromatin recruitment in HBV replicating cells has defined a broad repertoire cellular genes (~4.000) of and ncRNAs (75 miRNAs and 34 lncRNAs) potentially regulated by HBx with an enrichment in genes/ncRNAs involved in cell metabolism, chromatin dynamics and cancer but also in genes/ncRNAs that modulate HBV replication. HBc has also been shown to bind to cellular chromatin (~1000 genes) and to the promoters of a subset of cellular genes involved in inflammatory responses and innate immunity. Core protein (Cp) represents an attractive new therapeutic specific target for HBV chronic infection. Cp is essential for HBV genome packaging, reverse transcription, intracellular trafficking and the re-import of encapsidated HBV genomes into the nucleus, but due to its nuclear functions drugs targeting the Cp may also impact on cccDNA transcriptional activity and host genes reprogramming with a great potential for enhanced antiviral activity. The elucidation of the HBc crystal structure and the clarification of core dimers assembly process has led to the development of several compounds that target Cp and capsid assembly inhibit HBV replication therapeutic potential. We focused on two different classes of Cp assembly modulators. Heteroaryldihydropyrimidines (HAPs) act as allosteric effectors to increase the kinetics of assembly, strengthen dimer-dimer association and prevent the proper formation of viral capsids with the formation of aberrant core particles at high concentrations. The phenyl-propenamide derivatives AT-61 and AT-130 increase capsid assembly reaction rate, interfere with HBV RNA packaging and to the formation of apparently normal empty capsids. Interestingly, compounds belonging to the HAP chemical class have moved into clinical trials. In this third year of my PhD I further investigated the interplay between the impact of HAP12 and AT-130 treatment on viral replication and HBV core recruitment on the cccDNA and I expanded these observation on the regulation of cellular genes targeted by Cp. Methods I made use of three cellular models of HBV infection/replication: a) HepAD38 cells, a stable clone derived from the hepatocellular cell line HepG2, carry a complete integrated HBV genome under the control of a tetracycline- off inducible promoter; a HepG2-NTCP stable clone expressing high levels of the HBV entry receptor Na+/taurocholate co-transporting polypeptide (NTCP), that allow HBV infection in vitro; primary human hepatocytes (PHHs) from multiple donors infeted with HBV inocula purified from the cell supernatants of HepAD38 stable cell lines. Cells were treated with the anti-capsid HAP12 and AT-130 respectively 1 and 5 μM concentration to assess the effects on viral replication. All relevant virological parameters were evaluated: capsid-associated HBV-DNA (TaqMan real-time PCR); cccDNA levels (TaqMan real-time PCR); pgRNA levels (quantitative real-time PCR). Nuclear cccDNA was visualized by DNA FISH using using HBV specific probes encompassing the whole HBV genome. Anti-HBc, anti-HBx and anti-AcH4 ChIPs and cccDNA-ChIP experiments were performed in TET-released HepAD38 cells and in mock and HBV-infected NTCP-HepG2 cells and PHHs and analyzed by TaqMan real-time PCR using cccDNA and gene specific primers. Results I have shown that HAP12 and AT130, in addition to suppress efficiently HBV replication prevents the accumulation of nuclear cccDNA by blocking the recycling of mature core particles into the nucleus in TET released HepAD38 cells. Similarly, HAP12 treatment of NTCP-HepG2 cells at the time of HBV infection results in a drastic reduction of cccDNA formation, suggesting an important role of Cp for rcDNA release into the nucleus, conversion of rcDNA into cccDNA or cccDNA chromatinization. Conversely, when HAP12 treatment was started 10 days post-infection, when the cccDNA pool is established and stable in HBV infected NTCP-HepG2 cells, I observed very little or no effect on cccDNA levels, a significant reduction of cccDNA transcription and pgRNA levels together with a very strong inhibition of total HBV DNA and viral replication. These results indicate that HAP12, in addition to target capsid formation and pgRNA encapsidation in the cytoplasm, also affect cccDNA function in the nucleus. Next, I confirmed that Cp binds to the cccDNA in all the HBV infection/replication models and I showed that Cp recruitment on the cccDNA occurs as early as 2 hours post-infection in HBV infected PHHs, preceding the binding of HBx onto the cccDNA that becomes evident at 4-8 hours post-infection. Finally, I investigated the ability of Cp to bind to selected cellular promoters in HBV infected cells and the HAP12 onto Cp recruitment on these promoters. To this aim I performed anti-HBc ChIP assays in TET released HepAD38 cells, HBV infected NTCP-HepG2 cells and HBV infected PHHs and I confirmed Cp binding to the regulatory regions of the Ezh2 histon methyl-transferase, the cSrc proto-oncogene (that has been also shown to potentiate HBV replication), the E2F1 transcription factor and cell cycle regulator, and the IL29/lamda3 interferon, whereas the IL6 promoter was consitently not enriched and served as negative control. Importantly, HAP treatment was able to blocks Cp recruitment on genes promoters. Altogether these results identify capsid inhibitors as the first class of “virus specific” compounds capable to target the cccDNA functions and potentially counteract Cp pathogenicity in infected hepatocytes

EZH2, JMJD3 and UTX epigenetically regulate hepatic plasticity inducing retro-differentiation and proliferation of liver cells

Modification of histones by lysine methylation plays a role in many biological processes, and it is dynamically regulated by several histone methyltransferases and demethylases. The polycomb repressive complex contains the H3K27 methyltransferase EZH2 and controls dimethylation and trimethylation of H3K27 (H3K27me2/3), which trigger gene suppression. JMJD3 and UTX have been identified as H3K27 demethylases that catalyze the demethylation of H3K27me2/3, which in turns lead to gene transcriptional activation. EZH2, JMJD3 and UTX have been extensively studied for their involvement in development, immune system, neurodegenerative disease, and cancer. However, their role in molecular mechanisms underlying the differentiation process of hepatic cells is yet to be elucidated. Here, we show that EZH2 methyltransferase and JMJD3/UTX demethylases were deregulated during hepatic differentiation of human HepaRG cells resulting in a strong reduction of H3K27 methylation levels. Inhibition of JMJD3 and UTX H3K27 demethylase activity by GSK-J4 epi-drug reverted phenotype of HepaRG DMSO-differentiated cells and human primary hepatocytes, drastically decreasing expression of hepatic markers and inducing cell proliferation. In parallel, inhibition of EZH2 H3K27me3 activity by GSK-126 epi-drug induced upregulation of hepatic markers and downregulated the expression of cell cycle inhibitor genes. To conclude, we demonstrated that modulation of H3K27 methylation by inhibiting methyl-transferase and dimethyl-transferase activity influences the differentiation status of hepatic cells, identifying a possible new role of EZH2, JMJD3 and UTX epi-drugs to modulate hepatic cell plasticity

Nicotine Changes Airway Epithelial Phenotype and May Increase the SARS-COV-2 Infection Severity

(1) Background: Nicotine is implicated in the SARS-COV-2 infection through activation of the α7-nAChR and over-expression of ACE2. Our objective was to clarify the role of nicotine in SARS-CoV-2 infection exploring its molecular and cellular activity. (2) Methods: HBEpC or si-mRNA-α7-HBEpC were treated for 1 h, 48 h or continuously with 10−7 M nicotine, a concentration mimicking human exposure to a cigarette. Cell viability and proliferation were evaluated by trypan blue dye exclusion and cell counting, migration by cell migration assay, senescence by SA-β-Gal activity, and anchorage-independent growth by cloning in soft agar. Expression of Ki67, p53/phospho-p53, VEGF, EGFR/pEGFR, phospho-p38, intracellular Ca2+, ATP and EMT were evaluated by ELISA and/or Western blotting. (3) Results: nicotine induced through α7-nAChR (i) increase in cell viability, (ii) cell proliferation, (iii) Ki67 over-expression, (iv) phospho-p38 up-regulation, (v) EGFR/pEGFR over-expression, (vi) increase in basal Ca2+ concentration, (vii) reduction of ATP production, (viii) decreased level of p53/phospho-p53, (ix) delayed senescence, (x) VEGF increase, (xi) EMT and consequent (xii) enhanced migration, and (xiii) ability to grow independently of the substrate. (4) Conclusions: Based on our results and on evidence showing that nicotine potentiates viral infection, it is likely that nicotine is involved in SARS-CoV-2 infection and severity

{DNA} repair protein {DNA}-{PK} protects {PC}12 cells from oxidative stress-induced apoptosis involving {AKT} phosphorylation

Background Emerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair.Methods and results We used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of gamma Eta 2 Alpha X foci, detected by immunofluorescence, and gamma H2AX levels detected by western blot analysis. After 24 h of cell recovery, gamma Eta 2 Alpha X foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage.Conclusions Taken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme

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

Smart housing and mobility for the third age Progetto S.I.A.M.A.D.A

In occasione del centenario della nascita del Bauhaus, la SID ha dedicato l'Assemblea annuale a una riflessione sulla ricerca di design, ristabilendo un dialogo con la Scuola che è stata il primo modello pedagogico e didattico. Partendo anche dal contributo essenziale al rinnovamento dell'eredità del Bauhaus fornito da Tomás Maldonado, sono state individuate tre questioni aperte, che ancora oggi rappresentano quadri di riferimento scientifico e culturale delle ricerche in design: 1. Design e altri saperi; 2. Design e sperimentazione; 3. Design e identità di genere. La riflessione ha raccolto i contributi dei docenti e dei ricercatori della comunità scientifica proposti come progetti di ricerca (in corso o conclusi da non più di due anni) e come Idee di ricerca inedite (presentate da giovani under 40). La cura editoriale ha portato ad un lavoro di analisi e mappatura che propone un panorama della ricerca in design svolta nelle Università italiane

Detection of Pathological Markers of Neurodegenerative Diseases following Microfluidic Direct Conversion of Patient Fibroblasts into Neurons

Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are clinically diagnosed using neuropsychological and cognitive tests, expensive neuroimaging-based approaches (MRI and PET) and invasive and time-consuming lumbar puncture for cerebrospinal fluid (CSF) sample collection to detect biomarkers. Thus, a rapid, simple and cost-effective approach to more easily access fluids and tissues is in great need. Here, we exploit the chemical direct reprogramming of patient skin fibroblasts into neurons (chemically induced neurons, ciNs) as a novel strategy for the rapid detection of different pathological markers of neurodegenerative diseases. We found that FAD fibroblasts have a reduced efficiency of reprogramming, and converted ciNs show a less complex neuronal network. In addition, ciNs from patients show misfolded protein accumulation and mitochondria ultrastructural abnormalities, biomarkers commonly associated with neurodegeneration. Moreover, for the first time, we show that microfluidic technology, in combination with chemical reprogramming, enables on-chip examination of disease pathological processes and may have important applications in diagnosis. In conclusion, ciNs on microfluidic devices represent a small-scale, non-invasive and cost-effective high-throughput tool for protein misfolding disease diagnosis and may be useful for new biomarker discovery, disease mechanism studies and design of personalised therapies

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