The Liver Diseases Virology Section focuses on understanding the mechanisms of disease and to improve treatment and prevention of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, and viral hepatitis-associated hepatocellular carcinoma (HCC).
Hepatitis B and C viruses together infect more than 10 percent of the world population and are the most common causes of chronic liver disease and hepatocellular carcinoma, which is now the fourth leading cause of death from cancer in the world1. Current therapies for HBV are less than optimal. Recent advances of direct-acting antivirals in HCV therapy have substantially improved the outlook for millions of HCV-infected people. The prospect for a HCV vaccine remains elusive. Given the global public health burden, it is crucial to improve on strategies to control the two infections including more affordable and effective therapies and preventive measures. To achieve this goal, we need to understand viral life cycle, viral mechanisms of productive and persistent infection, and virus-host interactions at the molecular, cellular and genomic levels. The thematic effort of my laboratory focuses on these topics.
While many gaps in our knowledge of HCV still exist, great strides have been made in characterizing the virus and functions of viral genes as well as in unraveling the replication pathway and immunologic mechanisms of liver injury. HCV, like many other viruses, exploits host cellular machinery for productive infection. However, unlike other RNA viruses, HCV has a high propensity to cause persistent infection despite an active host immune response. Thus, it commands unique mechanisms to counteract the various host defenses. HCV gene products have been shown to interact with many host factors and to induce cellular alterations vital for viral replication, persistence and pathogenesis. The development of in vitro systems including infectious cell cultures affords the opportunity to fully characterize viral replication and virus-cell interactions as well as developing novel therapeutics. SCID/uPA mice transplanted with human hepatocytes susceptible to HCV infection and transgenic mouse models expressing human HCV entry factors are useful small animal models.
For HBV, replication and virus-host interactions have been studied extensively. The availability of HBV culture systems and small animal models has greatly advanced fundamental knowledge of the virus and the evaluation of vaccine and therapeutic candidates. However, the complexity of virus-host interactions during HBV infection remains unclear. In addition, chronic infection with both viruses is associated with development of hepatocellular carcinoma (HCC) that carries high morbidity and mortality and is difficult to diagnose and treat. Virus-associated HCC remains poorly understood and is an important unmet need in liver diseases.
- To study virus-host interactions by applying genome-wide technologies and systems biology approaches.
- To apply and develop cutting-edge technologies and model systems to discover novel small molecule probes of HBV and HCV life cycles and to explore them for potential therapeutic advances.
- To investigate the mechanisms of action of antiviral therapy and to explore the role of innate immunity in viral pathogenesis and treatment response.
We successful adopted the technology of iPSC and hepatocyte differentiation as in vitro and in vivo models to study HBV and HCV infections. We demonstrated that these differentiated, hepatocyte-like cells (HLCs), despite an immature phenotype, are permissive to HCV infection in vitro and mount an interferon response. The HLCs could be engrafted in the liver parenchyma of immunodeficient mice. The HLCs were maintained for more than 3 months in the liver of the chimeric mouse, where they underwent further maturation and proliferation. These chimeric mice were permissive to infection with HBV and HCV-positive sera of different genotypes and support long-term infection.
Finally, we have established an active research program on the increasingly important problem of hepatocellular carcinoma in the context of chronic viral hepatitis. We are developing various in vitro and in vivo models to identify and study potentially important procarcinogenic pathways that are triggered by HBV or HCV infection. By leveraging this knowledge, we hope to uncover novel strategies for preventive and therapeutic interventions of this deadly cancer.
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 Hepatits 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.