RNA Expression Profile of HBV-infected Primary Human Hepatocytes treatment by Interferon subtypes.
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ABSTRACT: Microarray-dependent transcriptional analysis of interferon stimulated genes treated with different kind of interferon subtypes in HBV-infected PHH.
Project description:Monitor HBV mRNA reduction in response to Tazarotene and to identify global transcriptome-wide gene expression associated with Tazarotene treatment in HBV infected PHH using RNA-Seq
Project description:Several classes of capsid assembly modulators (CAMs) are currently being developed for chronic hepatitis B (CHB) cure. Both class A (CAM-A) and class E (CAM-E) CAMs disrupt nucleocapsid assembly and reduce extracellular hepatitis B virus (HBV) DNA. However, only CAM-As have been shown to reduce the number of HBV-infected cells in the animal models. However, there has been limited efficacy to date of CAM-A molecules achieving this secondary mechanism of HBV-infected cell clearance in CHB clinical trials. To investigate this disconnect, we performed comparative experiments with tool compounds from each class to further explore these unique features and antiviral activity of CAM-A across HBV-infected primary human hepatocytes (PHH), as well as in two different HBV mouse models (immunodeficient mice repopulated with human hepatocytes and AAV-HBV). Mechanistic studies in HBV-infected PHH revealed that CAM-A, but not CAM-E, induced dose-dependent aggregation of HBV core protein (HBc) in the nucleus. Experiments with siRNA, resulted in identification of the ubiquitin-binding protein p62 as a factor negatively regulating the size of these aggregates. Furthermore, we found that only the CAM-A is able to induce HBc-positive cell death in vivo with the loss of HBV-infected cells positively correlated to the levels of intrahepatic HBc. Profiling of intrahepatic HBc levels across CHB patient liver biopsies demonstrated a significantly lower level of HBc per hepatocyte than either of the HBV mouse models. Taken together, these data demonstrate that CAMs of class A have a unique secondary mechanism that has potential to specifically affect viability of HBV-infected hepatocytes. At the same time, the clearance of infected hepatocytes may depend on the level of HBc expression thereby limiting the therapeutic potential for this class of molecules.
Project description:Cell: HBV-infected PHH cells. Methods: PHH cells were infected with 2000 genome equivalents/cell of HBV particles in the presence of 4% PEG8000. Seven days after HBV infection, ChIP-Seq analysis of cccDNA in HBV-infected PHHs was carried out. HBV-infected PHH cells were digested with micrococcal nuclease and resulting mononucleosomes purified by sucrose gradient centrifugation. Nucleosomes were enriched with anti-H3K79succ antibodies by ChIP assay and the associated DNA contained both human and HBV DNA fragments was analyzed by deep sequencing. The HBV-specific reads in ChIP-Seq pilot experiments was quantified and normalized by the reads mapped to the human genome.
Project description:Human primary hepatocytes isolated from chimeric mice were infected with HBV for 7 days. The comprehensive changes of miRNA levels were determined by miRNA array. MicroRNA expression levels were compared in between control and HBV-replicating primary hepatocytes by 2K microRNA microarrays