Biology subjects

Rivoire, M.

Publications and source records attributed to Rivoire, M..

7 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

An optimized model for HEV infection in the HepaRG cell line

Hepatitis E virus (HEV) causes acute hepatitis that can progress to fulminant or chronic hepatitis. For decades, the lack of a pertinent and robust cell culture system for HEV has delayed our understanding on this hepatotropic virus. HepaRG cells are one of the few hepatocyte-derived cell lines able to replicate HEV. These cells can differentiate (dHepaRG) into hepatocytes and cholangiocytes upon treatment with dimethyl sulfoxyde (DMSO) and are very relevant to study interactions between pathogens and hepatocyte innate immunity. However, the suitability of the HepaRG model to study HEV need to be further investigated. In this study, we found that HEV can infect proliferating HepaRG cells and that DMSO-induced differentiation is not necessary for HEV infection. Moreover, even if treatment with DMSO is needed to maintain optimal differentiation and polarization of dHepaRG, its presence is detrimental for HEV infection. Overall, this study shows that dHepaRG cells cultured without DMSO is a suitable model to study HEV and its interaction with the hepatocyte innate system.

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

THE DUAL-SPECIFICITY KINASE DYRK1A INTERACTS WITH THE HEPATITIS B VIRUS GENOME AND REGULATES THE PRODUCTION OF VIRAL RNA

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. Beside pegylated interferon-alpha, there is currently no clinically approved therapeutic protocol able to target cccDNA [1]. The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins (RBPs), could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program.

microbiology↗

bioRxiv2024

Deciphering hepatitis B virus phospho-signature in primary human hepatocytes

Phosphorylation is a major post-translation modification (PTM) of proteins which is finely tuned by the activity of several hundred kinases and phosphatases. It controls most if not all cellular pathways including anti-viral responses. Accordingly, viruses often induce important changes in the phosphorylation of host factors that can either promote or counteract viral replication. Among more than 500 kinases constituting the human kinome only few have been described as important for the Hepatitis B virus (HBV) infectious cycle, and most of them intervene during early or late infectious steps by phosphorylating the viral Core protein (HBc) protein. In addition, little is known on the consequences of HBV infection on the activity of cellular kinases. The objective of this study was to investigate the global impact of HBV infection on the cellular phosphorylation landscape early after infection. For this, primary human hepatocytes (PHHs) were challenged or not with HBV, and a mass spectrometry (MS)-based quantitative phosphoproteomic analysis was conducted two- and seven-days post-infection. The results indicated that while, as expected, HBV infection only minimally modified the cell proteome, significant changes were observed in the phosphorylation state of several host proteins at both times points. Gene enrichment and ontology analyses of up- and down-phosphorylated proteins revealed common and distinct signatures induced by infection. In particular, HBV infection resulted in up-phosphorylation of proteins involved in DNA damage signaling and repair, RNA metabolism, in particular splicing, and cytoplasmic cell-signaling. Down-phosphorylated proteins were mostly involved in cell signaling and communication. Validation studies carried out on selected up-phosphorylated proteins, revealed that HBV infection induced a DNA damage response characterized by the appearance of 53BP1 foci, the inactivation of which by siRNA increased cccDNA levels. In addition, among up-phosphorylated RNA binding proteins (RBPs), SRRM2, a major scaffold of nuclear speckles behaved as an antiviral factor. In accordance with these findings, kinase prediction analysis indicated that HBV infection upregulates the activity of major kinases involved in DNA repair. These results strongly suggest that HBV infection triggers an intrinsic anti-viral response involving DNA repair factors and RBPs that contribute to reduce HBV replication in cell culture models.

microbiology↗

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↗

bioRxiv2020

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies. Author SummaryChronic infection with Hepatitis B virus (HBV) affects more than 250 millions of people world-wide and is a major global cause of liver cancer. Current treatments lead to a significant reduction of viremia in patients. However, viral clearance is rarely obtained and the persistence of the HBV genome in the hepatocytes nucleus generates a stable source of viral RNAs and subsequently proteins which play important roles in immune escape mechanisms and liver disease progression. Therapies aiming at efficiently and durably eliminating viral gene expression are still required. In this study, we identified the nuclear partners of the HBV Core protein (HBc) to understand how this structural protein, responsible for capsid assembly in the cytoplasm, could also regulate viral gene expression. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs). One of these RBPs, SRSF10, was demonstrated to restrict HBV RNA levels and a drug, able to alter its phosphorylation, behaved as an antiviral compound capable of reducing viral gene expression. Altogether, this study sheds new light novel regulatory functions of HBc and provides information relevant for the development of antiviral strategies aiming at preventing viral gene expression.

microbiology↗