Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes.

BACKGROUND & AIMS: Transcription termination fine tunes gene expression and contributes to specify the function of RNAs in eukaryotic cells. Transcription termination of hepatitis B virus (HBV) is subjected to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. The regulation of the usage of this cPAS and its impact on viral gene expression and replication is currently unknown. APPROACH & RESULTS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE-PCR assay coupled to single molecule sequencing both in in vitro infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated to HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants for HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: Hepatitis B virus (HBV) covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail to be attained to achieve HBV cure. Transcriptional fidelity is a cornerstone in gene expression regulation. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit the recognition of HBV polyadenylation signal and transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in HBV chronically infected patients suggests a role for the helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

Virus hijacking of host epigenetic machinery to impair immune response.

Epigenetic modifications, such as DNA hypermethylation, histone acetylation/methylation, or nucleosome positioning, result in differential gene expression. These modifications can have an impact on various pathways, including host antiviral immune responses. In this review, we summarize the current understanding of epigenetic modifications induced by viruses to counteract host antiviral immune responses, which are crucial for establishing and maintaining infection of viruses. Finally, we provide insights into the potential use of epigenetic modulators in combating viral infections and virus-induced diseases.

DNA damage reduces heterogeneity and coherence of chromatin motions.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

HIRA Supports Hepatitis B Virus Minichromosome Establishment and Transcriptional Activity in Infected Hepatocytes.

BACKGROUND & AIMS: Upon hepatitis B virus (HBV) infection, partially double-stranded viral DNA converts into a covalently closed circular chromatinized episomal structure (cccDNA). This form represents the long-lived genomic reservoir responsible for viral persistence in the infected liver. Although the involvement of host cell DNA damage response in cccDNA formation has been established, this work investigated the yet-to-be-identified histone dynamics on cccDNA during early phases of infection in human hepatocytes. METHODS: Detailed studies of host chromatin-associated factors were performed in cell culture models of natural infection (ie, Na+-taurocholate cotransporting polypeptide (NTCP)-overexpressing HepG2 cells, HepG2hNTCP) and primary human hepatocytes infected with HBV, by cccDNA-specific chromatin immunoprecipitation and loss-of-function experiments during early kinetics of viral minichromosome establishment and onset of viral transcription. RESULTS: Our results show that cccDNA formation requires the deposition of the histone variant H3.3 via the histone regulator A (HIRA)-dependent pathway. This occurs simultaneously with repair of the cccDNA precursor and independently from de novo viral protein expression. Moreover, H3.3 in its S31 phosphorylated form appears to be the preferential H3 variant found on transcriptionally active cccDNA in infected cultured cells and human livers. HIRA depletion after cccDNA pool establishment showed that HIRA recruitment is required for viral transcription and RNA production. CONCLUSIONS: Altogether, we show a crucial role for HIRA in the interplay between HBV genome and host cellular machinery to ensure the formation and active transcription of the viral minichromosome in infected hepatocytes.

Characterization and implementation of a miniature X-ray system for live cell microscopy.

The study of radiation effects on biological tissues is a diverse field of research with direct applications to improve human health, in particular in the contexts of radiation therapy and space exploration. Understanding the DNA damage response following radiation exposure, which is a key determinant for mutagenesis, requires reproducible methods for delivering known doses of ionizing radiation (IR) in a controlled environment. Multiple IR sources, including research X-ray and gamma-ray irradiators are routinely used in basic and translational research with cell and animal models. These systems are however not ideal when a high temporal resolution is needed, for example to study early DNA damage responses with live cell microscopy. Here, we characterize the dose rate and beam properties of a commercial, miniature, affordable, and versatile X-ray source (Mini-X). We describe how to use Mini-X on the stage of a fluorescence microscope to deliver high IR dose rates (up to 29 Gy/min) or lower dose rates (≤ 0.1 Gy/min) in live cell imaging experiments. This article provides a blueprint for radiation biology applications with high temporal resolution, with a step-by-step guide to implement a miniature X-ray system on an imaging platform, and the information needed to characterize the system.

Performance of deep learning restoration methods for the extraction of particle dynamics in noisy microscopy image sequences.

Particle tracking in living systems requires low light exposure and short exposure times to avoid phototoxicity and photobleaching and to fully capture particle motion with high-speed imaging. Low-excitation light comes at the expense of tracking accuracy. Image restoration methods based on deep learning dramatically improve the signal-to-noise ratio in low-exposure data sets, qualitatively improving the images. However, it is not clear whether images generated by these methods yield accurate quantitative measurements such as diffusion parameters in (single) particle tracking experiments. Here, we evaluate the performance of two popular deep learning denoising software packages for particle tracking, using synthetic data sets and movies of diffusing chromatin as biological examples. With synthetic data, both supervised and unsupervised deep learning restored particle motions with high accuracy in two-dimensional data sets, whereas artifacts were introduced by the denoisers in three-dimensional data sets. Experimentally, we found that, while both supervised and unsupervised approaches improved tracking results compared with the original noisy images, supervised learning generally outperformed the unsupervised approach. We find that nicer-looking image sequences are not synonymous with more precise tracking results and highlight that deep learning algorithms can produce deceiving artifacts with extremely noisy images. Finally, we address the challenge of selecting parameters to train convolutional neural networks by implementing a frugal Bayesian optimizer that rapidly explores multidimensional parameter spaces, identifying networks yielding optimal particle tracking accuracy. Our study provides quantitative outcome measures of image restoration using deep learning. We anticipate broad application of this approach to critically evaluate artificial intelligence solutions for quantitative microscopy.

Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients.

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B.

Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus.

In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were type-I IFNs, IFNγ, type-III IFNs, TNFα, IL-6, IL-1ß, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1ß, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1ß and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1ß was found to have a very potent antiviral effect against HBV in vitro. HBV was previously shown to promptly inhibit IL-1ß production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1ß may help to further inhibit HBV replication in vivo.

Targeted glomerular angiopoietin-1 therapy for early diabetic kidney disease.

Vascular growth factors play an important role in maintaining the structure and integrity of the glomerular filtration barrier. In healthy adult glomeruli, the proendothelial survival factors vascular endothelial growth factor-A (VEGF-A) and angiopoietin-1 are constitutively expressed in glomerular podocyte epithelia. We demonstrate that this milieu of vascular growth factors is altered in streptozotocin-induced type 1 diabetic mice, with decreased angiopoietin-1 levels, VEGF-A upregulation, decreased soluble VEGF receptor-1 (VEGFR1), and increased VEGFR2 phosphorylation. This was accompanied by marked albuminuria, nephromegaly, hyperfiltration, glomerular ultrastructural alterations, and aberrant angiogenesis. We subsequently hypothesized that restoration of angiopoietin-1 expression within glomeruli might ameliorate manifestations of early diabetic glomerulopathy. Podocyte-specific inducible repletion of angiopoietin-1 in diabetic mice caused a 70% reduction of albuminuria and prevented diabetes-induced glomerular endothelial cell proliferation; hyperfiltration and renal morphology were unchanged. Furthermore, angiopoietin-1 repletion in diabetic mice increased Tie-2 phosphorylation, elevated soluble VEGFR1, and was paralleled by a decrease in VEGFR2 phosphorylation and increased endothelial nitric oxide synthase Ser(1177) phosphorylation. Diabetes-induced nephrin phosphorylation was also reduced in mice with angiopoietin-1 repletion. In conclusion, targeted angiopoietin-1 therapy shows promise as a renoprotective tool in the early stages of diabetic kidney disease.