Biology subjects

Levrero, M.

Publications and source records attributed to Levrero, M..

4 recordsLinked to original sources

bioRxiv2024

Identification of Cyclin L1 as a host factor regulating Hepatitis B Virus replication

Background & AimsUnderstanding the regulatory interactions between Hepatitis B virus (HBV) and human host factors is key to the development of next-generation host-directed antiviral therapies and achieving a functional HBV cure. In this study, we aimed to investigate HBV-induced alterations in host gene expression in primary human hepatocytes (PHH) to identify host-specific factors that are regulated and exploited by the virus for replication and survival. MethodsWe performed whole transcriptome sequencing (WTS) of HBV-infected PHH to identify host pathways that could potentially influence the HBV life cycle. RNA-interference-based validation of putative targets to evaluate the function of dysregulated candidate genes resulted in the identification of Cyclin L1 (CCNL1) as a key host factor. ResultsRNAi-knockdown of CCNL1 revealed that it is essential for HBV gene expression, including HBV-surface antigen (HBsAg). Mechanistically, we found that CCNL1 can phosphorylate the C-terminal domain (CTD) of RNA Polymerase II (RNAPII) at serine 2 (S2), likely to regulate HBV transcription. Furthermore, the knockdown of CCNL1 inhibited the binding of total and phospho- (Ser2-Ser5) RNAPII, pan-acetylated H3ac, and H3K27ac to HBV cccDNA, implicating its function in the regulation of cccDNA-dependent viral transcription. Finally, enhanced CCNL1 expression in chronic hepatitis B patients, as compared to those with resolved infection, underscores a functional link between this host factor and CHB. ConclusionOur data demonstrates that CCNL1 regulates HBV RNA transcription and replication by modulating RNAPII phosphorylation and activity, making it a potential host susceptibility factor for HBV. LAY SUMMARYHepatitis B requires human host cell factors and biological processes to establish an efficient infection. Identifying host factors that support and/or restrict HBV infection is essential for understanding the molecular basis of chronic HBV infection and for developing host-targeting anti-HBV drugs. Here, we report that CCNL1 can serve as a potential host susceptibility factor for HBV, as reduced CCNL1 function results in reduced viral replication and gene expression. Graphical SummaryGraphical summary, highlighting the approach and validation experiments. From whole transcriptomics analysis, we identified known HBV-host factors such as SRPK1, CDK1, NXF1 among others as well as new factors such as CCNL1. Employing various RNAi approaches and different cell models including primary human hepatocytes, we validated the role of CCNL1 during HBV infection cycle. Pull-down experiments followed by ChIP-PCR further showed a reduction in cccDNA-based transcription upon knockdown of CCNL1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/619969v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1ee9cd9org.highwire.dtl.DTLVardef@7bac2dorg.highwire.dtl.DTLVardef@164c3bborg.highwire.dtl.DTLVardef@6e497a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

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↗

bioRxiv2022

RNA helicase DDX5 mediates adaptive response to multi-kinase inhibitors in liver cancer

ObjectiveTo determine the role of RNA helicase DDX5 in sorafenib/multi-tyrosine kinase inhibitor (mTKI) response. Sorafenib and mTKIs downregulate DDX5 in vitro and preclinical hepatocellular carcinoma (HCC) models. In turn, sorafenib-mediated DDX5 downregulation activates Wnt/{beta}-catenin and non-canonical NF-{kappa}B signaling, resulting in ferroptosis escape and mTKI resistance. Design and ResultsMolecular, pharmacologic and bioinformatic approaches were employed in human HCC cell lines, preclinical HCC models, and HCCs from TCGA. Earlier studies linked sorafenib effectiveness to ferroptosis. Herein we demonstrate sorafenib/mTKIs downregulate DDX5 in vitro and in vivo. To understand the effect of DDX5 downregulation, we compared TCGA-derived HCCs expressing low vs. high DDX5 focusing on ferroptosis-related genes. Glutathione Peroxidase 4 (GPX4), a key ferroptosis regulator, was significantly overexpressed in DDX5LOW HCCs. Importantly, DDX5-knockdown (DDX5KD) HCC cell lines lacked lipid peroxidation by GPX4 inhibition, indicating DDX5 downregulation suppresses ferroptosis. RNAseq of wild type vs. DDX5KD cells untreated or treated with sorafenib, identified a unique set of genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps genes from Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, including NF-{kappa}B inducing Kinase required for non-canonical NF-{kappa}B activation. Pharmacologic inhibition of these pathways in combination with sorafenib reduced DDX5KD cell viability. Mechanistically, sorafenib-mediated NIK expression induced NRF2 transcription, while DDX5KD extended NRF2 half/life by stabilizing p62/SQSTM1, enhancing GPX4 expression and ferroptosis escape. ConclusionSorafenib/mTKI-mediated DDX5 downregulation results in adaptive mTKI resistance by enhancing NRF2 expression, leading to ferroptosis escape. We propose inhibition of the pathways leading to NRF2 expression will enhance the therapeutic effectiveness of sorafenib/mTKIs. O_LIWhat is already known on this topic - In advanced HCC, mTKIs/sorafenib, offer limited survival benefits due to resistance. Combination of VEGF (bevacizumab) and PD-L1 antibody (atezolizumab) has led to its adoption as first line treatment; however, mTKIs are still widely used in patients with contra-indications to bevacizumab; also, in atezolizumab/bevacizumab failure, mTKIs are the gold standard in second or later-line systemic therapy, emphasizing the importance of delineating the mechanism of mTKI resistance. C_LIO_LIWhat this study adds - We show mTKIs and sorafenib downregulate the RNA helicase DDX5. This downregulation of DDX5 by sorafenib enables activation of Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways, leading to expression of genes that enable escape from ferroptotic cell death. C_LIO_LIHow this study might affect research, practice or policy The Wnt/{beta}-catenin and non-canonical NF-{kappa}B pathways activated by sorafenib/DDX5 downregulation can serve as druggable targets to enhance the anti-cancer effect of mTKIs by inducing ferroptosis, thereby opening-up new therapeutic directions against mTKI resistance. C_LI

cancer biology↗

bioRxiv2022

Performance of the cobas(R) HBV RNA Automated Investigational Assay for the Detection and Quantification of Circulating HBV RNA in Chronic HBV Patients

BackgroundThe amount of HBV RNA in peripheral blood may reflect HBV covalently closed circular DNA (cccDNA) transcriptional activity within infected hepatocytes. Quantification of circulating HBV RNA (cirB-RNA) is thus a promising biomarker for monitoring antiviral treatment. ObjectivesWe evaluated the performance of an automated, prototype quantitative HBV RNA assay for use on the Roche cobas(R) 6800/8800 systems. Study DesignThe sensitivity, specificity, linearity, and potential interference by HBV DNA of the cobas(R) HBV RNA assay were assessed using synthetic HBV armored RNA and clinical specimens. Resultscobas(R) HBV RNA results were linear between 10 and 107 copies/mL in clinical samples of several HBV genotypes, and up to 109 copies/mL with synthetic RNA. Precision and reproducibility were excellent, with standard deviation below 0.15 log10 copies/mL and coefficients of variation below 5% throughout the linear range. The presence of HBV DNA had minimal (<0.3 log10 copies/mL) impact on HBV RNA quantification at DNA:RNA ratios of up to approximately one million. In a panel of 36 untreated patient samples, cirB-RNA concentrations were approximately 200-fold lower than HBV DNA. cirB-RNA was detected in all 13 HBeAg-positive patients (mean 6.0 log10 copies/mL), and in 20 of 23 HBeAg-negative patients (mean of quantifiable samples 2.2 log10 copies/mL). Finally, cirB-RNA was detected in 12 of 20 nucleoside analog-treated patients (mean of quantifiable samples 3.4 log10 copies/mL). ConclusionsThe cobas(R) 6800/8800 investigational HBV RNA assay is a high throughput, sensitive and inclusive assay to evaluate the clinical relevance of cirB-RNA quantification in patients with chronic hepatitis B.

microbiology↗