The research interests and current focus of the Clinical Research Section include: (1) natural history, management, genetics and treatment of viral hepatitis; (2) natural history, management, genetics and treatment of nonalcoholic steatohepatitis; (3) evaluation of the biology of non-cirrhotic portal hypertension; (4) study of hepatic involvement in other diseases such as Chronic Granulomatous Disease and Sickle Cell Disease; and (5) evaluation, management and therapy of primary biliary cirrhosis, autoimmune hepatitis and metabolic/genetic liver disease.
The LDB Clinical Research Section has an extensive biorepository of prospectively collected serum and DNA samples, and liver tissue from untreated and treated patients, with a variety of chronic liver conditions including chronic viral hepatitis (B, C and D), non-alcoholic fatty liver disease, non-cirrhotic portal hypertension, primary biliary cholangitis, primary sclerosing hepatitis, autoimmune hepatitis, Wilson’s disease and a variety of other less common chronic liver conditions, participating in natural history and investigator initiated clinical studies. The biorepository collection spans the period from the mid-1980s and continues to present day. These samples are available for clinical, translational and bench studies related to liver and other conditions upon request and approval from NIH.
Systems Biology of Host Factors in HCV Life Cycle
Using the complete HCV replication cycle (from entry to secretion) as a framework, all verified HCV host dependencies from this study were placed based on their predominant subcellular localization and relevance to particular stages of the viral life cycle. In addition, multiple datasets from other HCV siRNA screens and existing publications were mined, explored and integrated into a comprehensive up-to-date dataset of HCV interacting host factors. Computational mapping was performed to reconstitute the map that was further refined manually. HPFs (host proviral factors) are shown in red square, HAFs (host antiviral factors) are shown in green circle. Previously published HCV host dependencies that were also identified in this study are shown in yellow, and other known HCV host factors that were not identified in this study are shown in orange (HPF in circle and HAF in square).
HCV Infection Interferon Response and Lipid Droplet Biogenesis
A proposed model of innate antiviral response and HCV-induced lipogenesis and LD formation in HCV assembly. HCV can activate two distinct pathways. One is the induction of interferon pathway by interaction of HCV PAMP with RIG-I helicase like receptor (pattern recognition receptor) and the other is activation of lipogenic pathway via stress granule, DDX3X and Ikkα, that facilitates HCV assembly.
HCV Infection I-SMAD and Lipid Metabolism
A proposed model for I-SMAD-regulated signaling that enhances HSPG expression, cholesterol uptake and HCV entry. HCV entry is mediated by viral binding to HSPGs, LDLR and SR-BI (and other entry factors, not shown) on the host cell surface, thereby triggering a cassette of signaling to facilitate the internalization of HCV virions. HCV infection also induces the expression of SMAD6 and SMAD7, two I-SMADs of the TGF-β signaling pathway via NF-κB regulation. The I-SMADs translocate to the nucleus and transcriptionally activate the expression HSPG core protein and other cholesterol uptake receptors. Increased expression level of heparin sulfate on the cell surface in turn enhances HCV binding. Additionally, the I-SMADs and HSPGs can be induced by BMP6 and BMP7, the ligands in the BMP/TGF-β pathway.
Schematic of HBV replication and drug targets
The virus enters the hepatocyte using the sodium taurocholate cotransporting polypeptide as a receptor. cccDNA is generated by repairing the partially double stranded genome in the nucleus, whereupon viral transcription occurs. Encapsidation of the pregenomic RNA occurs in the cytoplasm via a complex interaction of viral and host proteins. Reverse transcription leading to negative and then positive strand synthesis occurs within the viral nucleocapsid. Viral assembly takes place in the ER. “Mature” nucleocapsids undergo assembly and coating with envelope proteins followed by budding and virion secretion into the blood. Potential targets for drug development are highlighted.
3-D structure of HCV
The three-dimensional structure of HCV is visualized and simulated by using electron cryomicroscopy of recombinant HCV-like viral particles.
iPSC-derived human hepatocytes
Human hepatocytes, generated from induced pluripotent stem cells derived from a patient, produce and exhibit hepatocyte-specific proteins (albumin) and functions (lipid and glycogen storage, organic anion transport). This regenerative medicine technology can be a valuable strategy for cell-based therapy of liver diseases.
Induction of steatosis by HCV
HCV infection of human hepatocyte-derived cells induces a lipogenic program that results in massive accumulation of lipid droplets (green and yellow structures). This is known as steatosis and is essential for HCV propagation.
Electron Micrograph of HCV-like Particles 1
Recombinant HCV-like particles produced in baculovirus-insect cell system are visualized by immunogold labeling in electron micrograph
Electron Micrograph of HCV-like Particles 2
Recombinant HCV-like particles generated in baculovirus-insect cells are purified and visualized by electron micrography.
Lipidation of Hepatitis C virus by Cellular Protein TM6SF2
During HCV budding inside the ER lumen, apolipoproteins B and E associate with the enveloped virus. Once released into the ER, the nascent virion acquires all the components of lipoprotein (apolipoproteins and lipids) to the infectious form of HCV. Together with MTP, TM6SF2 facilitates lipid loading onto the newly assembled virion during the budding and/or maturation process.
Spreading of HBV in Induced Hepatocyte-Like Cells
iHLCs were infected with HBV for 7 days, and then trypsinized and co-cultured with GFP-lentivirus transduced HLCs with or without Myrcludex B. Eight days after co-culture, RNAscope was used to evaluate HBV replication. White arrowheads indicate spreading of HBV to non-infected GFP-positive cells.
