Biology subjects

Grand, X.

Publications and source records attributed to Grand, X..

5 recordsLinked to original sources

bioRxiv2026

Adenine Base Editing Potently Suppresses Hepatitis B Surface Antigen Expression and Inhibits Hepatitis D Virus Release

Background and AimsNovel antiviral approaches capable of permanently inactivating the intrahepatic HBV DNA reservoir, the covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome, are urgently needed. This study evaluated adenine base editing as a strategy to disrupt HBV replication by introducing mutations in the overlapping HBs/polymerase open reading frame (ORF). MethodsAn adenine base editor (ABE) and 3 guide RNAs (gS1-gS3) were designed to introduce missense mutations within the HBs/polymerase ORF. ABE mRNA and individual gRNAs were co-transfected into HBV-infected HepG2-hNTCP cells and primary human hepatocytes. Antiviral efficacy was further assessed in HepG2.2.15 and PLC/PRF/5 cells harboring integrated HBV DNA. In vivo, lipid nanoparticles (LNP)-mediated delivery of ABE mRNA and gRNAs was evaluated in HBVcircle DNA-transduced mice and in HBV-infected human liver-chimeric mice. The impact of HBs editing on hepatitis D virus (HDV) release was assessed using PLC/PRF/5 and Huh7 cell-based HDV replication models. ResultsAdenine base editing efficiently reduced HBsAg production and HBV replication in vitro by targeting both cccDNA and integrated HBV DNA. A single LNP injection of ABE-gS2 resulted in undetectable HBsAg in HBVcircle mice, while two injections achieved a 90% reduction in serum HBsAg in HBV-infected human liver chimeric mice. HBV DNA replication was also inhibited in vivo. Furthermore, HBs ORF base editing markedly suppressed HDV release in vitro. ConclusionsAdenine base editing of the HBs ORF effectively impairs HBV replication and HBsAg production in vitro and in vivo and concomitantly inhibits HDV release, highlighting its therapeutic potential.

molecular biology↗

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

Bolero: a dedicated workflow to decipher Hepatitis B virus transcriptome from long-reads sequencing method coupled to 5RACE amplification of transcripts

Hepatitis B virus (HBV) represents a major health burden, as it affects close to 290 million people worldwide. Although prophylactic vaccines are available, current therapeutic compounds do not usually achieve HBV eradication due to the persistence of the covalently closed circular (ccc)DNA that serves as viral reservoir. Thus, novel biomarkers that reliably reflect intrahepatic cccDNA transcriptional activity would be highly relevant for the monitoring of infected individuals, as well as the evaluation of new treatments targeting HBV. In this context, the development of 5 rapid amplification of complementary DNA ends (5RACE) as a strategy to capture and amplify full-length HBV RNAs, coupled with long-read and full-length sequencing approaches (e.g., Oxford Nanopore Technology), has recently enabled the detailed characterization of these molecules. The analysis of such data requires a dedicated bioinformatics pipeline due to the highly condensed nature of the HBV genome, which is characterized by the production of multiple transcripts and spliced variants that overlap each other. Here, we present Bolero, a computational method and built-in workflow designed to handle HBV sequencing data and evaluate the relative expression of viral RNAs and their spliced variants. The analysis of HBV-infected cell lines demonstrates that our bioinformatics pipeline is efficient for the identification and quantification of individual HBV mRNAs. Thus, Bolero represents a useful tool to study cccDNA transcriptional activity and the heterogeneity of HBV RNA spliced variants. Author summaryTranscriptomic analyses have brought comprehensive insights in the mechanisms controlling gene expression. Moreover, with the recent advances in sequencing technologies and computational methods, researchers can nowadays not only quantify gene expression, but also study alternative splicing, polyadenylation, transcription initiation, and even rare phenomena such as distant gene fusions. However, conventional analysis tools still rely heavily on the assumption of linear genomes with minimal overlap between open reading frames, rendering them insufficient for studying complex viruses such as hepatitis B virus (HBV). Unlike typical linear genomes, HBV genome consists in a circular DNA molecule, which results in an extensive sequence overlap between its transcripts. To tackle these challenges, we developed an innovative approach coupling 5 rapid amplification of complementary DNA ends (5RACE) and long-read sequencing to comprehensively explore the HBV transcriptome. Furthermore, we developed Bolero, a computational method designed to handle the peculiarities of HBV sequencing data, which allows a detailed characterization of the HBV transcriptome.

bioinformatics↗

bioRxiv2024

The MYCN oncoprotein and helicases DDX17 and DDX5 have opposite effects on the production of chimeric transcripts in neuroblastoma cells

DEAD box helicases DDX17 and DDX5 control the termination of transcription and the associated cleavage of the 3 end of transcripts. Here we show that the transcriptional readthrough induced by their depletion in neuroblastoma cells also results in increased production of chimeric transcripts from tandemly oriented genes. Analysis of neuroblastoma tumours in which chimeric transcripts are abundant revealed that low expression of the DDX17 and DDX5 genes is associated with poor overall patient survival. Low DDX17 expression is also significantly associated with high-risk tumours and is inversely correlated with MYCN oncogene amplification, suggesting a link between these two factors. We demonstrate that changes in MYCN expression do not affect the expression of either helicase, but alter transcription termination leading to the production of chimeric transcripts. We provide evidence that MYCN acts on termination through its direct binding to the 3 region of genes and that it interacts with DDX17, suggesting that it may inhibit the activity of the helicase. Collectively, our work reveals a novel function of MYCN in transcription termination and suggests that the deregulation of MYCN and DDX17/DDX5 expression in neuroblastoma may lead to the expression of non-canonical and potentially harmful RNA molecules.

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↗