Biology subjects

Diederichs, A.

Publications and source records attributed to Diederichs, A..

4 recordsLinked to original sources

bioRxiv2025

Precision-cut liver slices as a model for the evaluation of host-targeting agents against hepatitis B and delta viruses

Background: Developing new therapeutic strategies against hepatitis B virus (HBV) and hepatitis delta virus (HDV) is essential to cure these infections. Yet evaluation of host-targeting agents (HTAs) with in vitro/in vivo models remains challenging. Therefore, we assessed precision-cut liver slices (PCLS) as an HBV/HDV co-infection model to study virus-host interactions and HTAs. Methods: We generated PCLS from human liver resections and infected them ex vivo with HBV and HDV. Tissue viability and architecture were monitored by intracellular ATP, secreted albumin, and multiplexed immunofluorescence. Viral markers were assessed by quantitative PCR, western blot, and light-sheet microscopy in infected slices following treatment with a sodium taurocholate co-transporting polypeptide (NTCP)-targeting peptide, lonafarnib, or the Toll-like receptor 8 agonist selgantolimod (SLGN). Single-cell RNA sequencing was performed to characterize the cellular responses to SLGN. Results: Here we show that our ex vivo infection protocol allows the establishment of HBV and HDV infection within the liver's three-dimensional architecture. Treatment with three well-characterized HTAs produced distinct antiviral effects consistent with each mechanism: an NTCP-targeting peptide blocked entry; lonafarnib induced intracellular hepatitis delta antigen accumulation; SLGN showed anti-HBV activity not previously seen in cultured hepatocytes, indicating cross-talk between immune and infected cells within liver slices. Conclusion: These results provide the characterization of PCLS as an ex vivo model of HBV/HDV co-infection and antiviral testing. In the future, PCLS could contribute to expediting pre-clinical characterization of HTAs and to exploring alternatives to animal experimentation.

microbiology↗

bioRxiv2025

DDX5 and DDX17 RNA helicases regulate hepatitis B virus RNA splicing

Chronic HBV infection remains a major health burden worldwide and is the main driver of severe liver diseases. Liver pathogenesis is associated with the increased proportion of HBV spliced variants that encode viral proteins involved in liver disease progression. However, how HBV RNA splicing is regulated is poorly understood. Here, we focused on DDX5 and DDX17 RNA helicases, known to regulate HBV RNA metabolism and alternative splicing of host genes. By performing 5RACE-PCR combined with single molecule sequencing, we demonstrated that silencing both proteins increased the usage of a specific splicing donor site and the expression of the derived HBV spliced variants. Polysome fractionation highlighted the ability of these RNA species to encode new viral proteins potentially contributing to liver pathogenesis. Overall, our data established DDX5 and DDX17 helicases as master regulators of HBV RNA metabolism, by fine-tuning viral splicing, which is linked to HBV-induced liver pathogenesis and disease progression.

molecular biology↗

bioRxiv2024

Helicases DDX5 and DDX17 promote Hepatitis B Virus transcription termination heterogeneity in infected human hepatocytes

Background & AimsTranscription 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 & ResultsTo 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. ConclusionsOur findings identify DDX5 and DDX17 as crucial determinants for HBV transcriptional fidelity and as host restriction factors for HBV replication.

molecular biology↗

bioRxiv2018

Interplay between the epigenetic enzyme lysine (K)-specific demethylase 2B and Epstein-Barr virus infection.

Histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in hematopoietic cells differentiation and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that KDM2B gene is differentially methylated in cell lines derived from the Epstein-Barr virus (EBV) associated endemic Burkitts lymphomas (eBL) compared to EBV negative sporadic BL cells. However, whether KDM2B plays a role in eBL development has never been previously demonstrated. Oncogenic viruses have been shown to hijack the host cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here we investigated whether EBV would alter KDM2B levels to enable its life cycle and promote B-cells transformation. We show that infection of B-cells with EBV leads to down-regulation of KDM2B levels. We also show that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV infected B-cells, we were able to show that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B-cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.\n\nIMPORTANCE. In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared to the sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared to sporadic Burkitt lymphomas cell lines, we identified several differential methylated genomic positions in proximity of genes with a potential role in cancer, among them the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has never been investigated before. In this study we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cells genes expression, thus highlighting a novel interaction between the virus and the cellular epigenome.

microbiology↗