Biology subjects

de Jong, Y. P.

Publications and source records attributed to de Jong, Y. P..

7 recordsLinked to original sources

bioRxiv2026

The HBV basal core promoter mutation confers a replicative advantage and transcriptionally reprograms hepatocytes toward HCC subtypes

Background: Hepatitis B virus (HBV) basal core promoter (BCP) and precore (PC) mutations occur during chronic HBV infection with the BCP-mutant being associated with increased hepatocellular carcinoma (HCC) risk. Objectives: The effects of these mutants on viral replication, hepatocyte biology, and carcinogenesis remain poorly defined. To address this, we characterized BCP- and PC-mutants using human hepatocyte chimeric mice and asked whether the resulting infection-induced transcriptomes correspond to subsets of human HBV-related hepatocellular carcinomas (HBV-HCCs). Design: Isogenic wild-type (WT), BCP- and PC-mutants of HBV genotypes A, C, and D (HBV-A, -C, -D) were generated from recombinant covalently-closed circular DNA to infect chimeric mice. HBV-HCC transcriptomic datasets from The Cancer Genome Atlas (TCGA) were used to stratify association of HBV variants with HCC subtypes. Results: WT, BCP-mutant, and PC-mutant HBV sequences remained genetically stable. The BCP mutation, but not the PC mutation, accelerated the rise in serum viremia in HBV-D- and HBV-A-infected chimeras; in HBV-C, acceleration required both (PC+BCP) mutations. Further comparisons of HBV-D variants revealed that the BCP-mutant increased intrahepatic viral DNA, viral protein expression, and upregulated cancer-related pathways, including transcripts associated with a subset of HBV-HCCs. Analysis of HBV-HCC samples from the TCGA revealed that tumors often harbor a mixture of WT and mutant transcripts, and that WT- and BCP-mutant-associated HCCs exhibit distinct transcriptional profiles. Conclusions: The HBV BCP-mutant directly perturbs hepatocyte homeostasis via virus-intrinsic mechanisms, selectively activating cancer-related pathways and defining a molecularly distinct subset of HBV-HCC. These findings suggest that HBV variants form distinct subcategories of HBV-HCCs.

molecular biology↗

bioRxiv2026

A CRISPR-Cas9 platform for primary human hepatocytes enables arrayed screening and in vivo validation of HBV host factors

More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.

genomics↗

bioRxiv2026

Orthogonal CRISPR screens and human liver chimeric mice identify hepatitis B virus host factors

Hepatitis B virus (HBV) chronically infects approximately 250 million people worldwide, and reliable curative therapies are lacking. A broader understanding of viral-host interactions could accelerate efforts to find new host-centric therapeutic targets. However, inefficient cell culture systems and limited replication markers compatible with pooled screening have precluded the widespread use of genetic perturbation screens. Here, we performed the first pooled, genome-wide CRISPR-Cas9 knockout (KO) screen with authentic HBV infection and integrated these results with two orthogonal pooled screens to identify host factors. We selected 72 genes for a multi-step assessment that included arrayed validation assays using both HBV infection and pgRNA transfection. We then independently tested thirteen genes using high-efficiency bulk KO experiments to guide further investigations of both antiviral and proviral factors. In both KO and siRNA-mediated knockdown experiments, depletion of the top antiviral factor, EXOC1, enhanced multiple HBV replication markers, and transcriptomic analysis revealed activation of hypoxia- and HIF-1 gene signatures. Three proviral factors, IRF2, WDR48, and ZCCHC14, were investigated in vivo using a human liver chimeric mouse model, which demonstrated that ZCCHC14 KO greatly reduced HBV replication and spread. Together, these complementary in vitro and in vivo platforms expand the catalog of HBV host factors and provide a scalable framework for host target discovery.

microbiology↗

bioRxiv2024

Ybx1 guides C/EBPα and cBAF chromatin-remodeling complex to promote adipogenic gene expression in steatotic hepatocytes

Excessive lipid accumulation by hepatocytes underlies the pathogenesis of metabolic-dysfunction associated steatotic liver disease (MASLD) and metabolic-dysfunction associated steatohepatitis (MASH) from the earliest stages of the disease. How liver cells regulate the commitment to storing large volumes of fat despite resulting tissue damage is not well understood. Here, we show Y box-binding protein 1 (Ybx1) is necessary for ectopic activation of an adipocyte-specific gene expression module that potentiates lipid accumulation in hepatocytes. Diet-induced obese (DIO) mice, with liver-specific depletion of Ybx1 (Ybx1LKO), are resistant to MASLD without becoming hyperlipidemic. Ybx1LKO livers exhibit upregulation of hepatocyte markers, like urea processing enzyme carbamoyl phosphate synthetase I (Cps1), and downregulation of adipocyte markers known to be transcriptionally regulated by peroxisome proliferator-activated receptor gamma (PPAR{gamma}). In nuclei of DIO mice, YBX1 interacts with CCAAT-enhancer-binding proteins alpha (C/EBP) and the canonical BRG1/BRM-associated factor complex (cBAF); and C/EBP is required for Ybx1-dependent PPAR{gamma} expression in cultured liver cells. The chromatin binding pattern of YBX1 from DIO mouse liver overlaps with those of C/EBP and cBAF at key adipogenic loci including Pparg and Cfd. However, most YBX1-DNA binding occurs on C/EBP-cBAF-depleted stretches located on chromosomes 16, 18, and 19, spanning up to five Mb, and overlapping regions which are inaccessible in differentiating preadipocytes, thereby bounding activational C/EBP-cBAF complex-DNA interactions. Moreover, YBX1 expression is increased up to nine-fold in the livers of obese patients with MASLD-MASH compared to healthy obese controls; and adipocyte-specific genes, upregulated by Ybx1, are also upregulated in human MASLD-MASH. Overall, our study uncovers Ybx1 as a critical epigenetic regulator in liver and potential therapeutic target for treatment of MASLD and MASH.

developmental biology↗

bioRxiv2020

Human hepatocyte PNPLA3 148M exacerbates rapid non-alcoholic steatohepatitis development in chimeric mice

Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to a hypercaloric Western-style diet. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused widespread microvesicular steatosis and even more severe steatohepatitis. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variants associated with advanced fatty liver diseases.

pathology↗

bioRxiv2020

Public broadly neutralizing antibodies against hepatitis B virus in individuals with elite serologic activity

SUMMARYAlthough there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and constitute clinical correlates of recovery from infection. To examine the human neutralizing antibody response to HBV in elite neutralizers we screened 144 individuals. The top individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection, but selected for resistance mutations in mice with established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with a peptide epitope containing residues frequently mutated in human immune escape variants revealed a loop anchored by oppositely charged residues. The structure provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.

immunology↗

bioRxiv2019

An RNA-based system to study hepatitis B virus replication and select drug-resistance mutations

Hepatitis B virus (HBV) chronically infects over 250 million people worldwide, increasing their risk of liver cirrhosis and hepatocellular carcinoma. There is a vaccine to prevent new infections, but no efficient cure for chronic infection. New insights into HBV biology are needed to improve cure rates for this widespread devastating disease. We describe a method to initiate replication of HBV, a DNA virus, using synthetic RNA. This approach has several advantages over existing systems: it eliminates contaminating background signal from input virus or plasmid DNA and can be easily adapted to multiple genotypes and mutants. Further, it can be applied to identify anti-HBV compounds, measure anti-HBV drug efficiency, study virus evolution, and, as we demonstrate, it can be uniquely applied to predict antiviral drug resistance.

microbiology↗