Molecular Mechanisms Of Hepatitis B Viral infection, Pathogenesis And Persistence

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

We have previously demonstrated that there are genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models. To further study genotype-specific genetic variations and pathogenesis, we studied two HBV subtypes of genotype A, A1 (commonly in Africa) and A2 (commonly in Europe). Individuals infected with these two subtypes appear to have different clinical manifestations and virologic features17. Using HBV generated from subtypes A1 and A2 in cell culture (HBVcc, as described above), we demonstrate that subtypes A1 and A2 can be passaged in vitro and in vivo and respond equally well to human IFN-α treatment. Subtype A2 showed a much higher viral replication level than that of subtype A1. Mechanistic investigations using constructs with chimeric A1/A2 sequences and specific mutations indicated that subtype A2 has an inherently higher replication phenotype due to specific polymorphisms in the HBx gene resulting in amino acid variations. Studies of HBx expression demonstrated that A1 HBx is expressed at a much lower level than that of A2 HBx. Mutagenesis studies identified two HBx amino acid variations responsible for the observed phenotypic difference. Using AlphaFold2, we generated structural models of HBx proteins of A1 and A2. Superposition of the two models reveal that the overall structural motifs are similarly aligned, except for the C-terminal peptides diverging between the A1 and A2 models, possibly explaining their functional difference. This genotypic difference potentially explains the reported clinical differences between the two subtypes as well as providing a previously unrecognized association between viral sequence variations and clinical manifestations of HBV infection in humans. HBV co-opts and interacts with an extensive array of host factors for productive infection. Herein, we apllied two distinct genome-wide approaches to identify and characterize these host factors. First, we developed an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host-dependency factors using flow cytometry. HepG2NTCP/Cas9 cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR and NTCP. Among the novel candidate genes, knock-out of 12 genes significantly decreased HBV replication markers. Validation using siRNA knock-down in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV pro-viral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication, likely by down-regulating CYP7A1 and bile acid metabolism. We thus identified previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development. In the second approach, we optimized and miniaturized the HBV-HepG2-NTCP infection system suitable for for a high-throughput plate-based AlphaLisa screen to detect viral antigen (HBc/eAg). An arrayed genome-wide screen targeting approximately 21,500 genes using three unique siRNA sequences per gene was performed using this platform. The validated candidate genes included previously identified HBV host factors, such as SLC10A1, HNF4A, CH25H, RXRA, PLK1 etc. Among the validated genes that were not known previously to be involved in HBV infection, nuclear receptor coactivator 5 (NCOA5) and chromodomain-helicase-DNA-binding protein 4 (CHDB4) were required for HBV replication as their knock-down (KD) led to reduced HBV replication. In contrast, neuroblastoma RAS viral oncogene homolog (NRAS) was shown to be an antiviral host factor. Mechanistically, NCOA5 likely operates via the estrogen receptor and hepatocyte nuclear factor 4 alpha (HNF4A) axis, while CHD4 modulates cccDNA histone modifications. NRAS knockdown enhanced HBV transcription by elevating HNF4A expression and inducing cell cycle arrest, which may explain the observed restriction of HBV replication in HBV-associated HCC tissues, where RAS hyperactivation is frequently observed, and the occurrence of HBV reactivation in chemotherapy targeting the RAS signaling pathway. This manuscript has provisionally been accepted in Journal of Hepatology. As illustrated by the two studies, the identification and characterization of host factors involved in the complete HBV life cycle will advance our understanding of HBV pathogenesis and uncover novel therapeutic targets.