The American Liver Foundation supports research for treatments and cures for liver disease. This section provides information about the current Hepatitis C research projects.
Genetic Dissection of Hepatitis C Virus Entry in Vivo
Alexander Ploss, PhD, Gregg Allman and Tune In to Hep C Liver Scholar Award winner
The Rockefeller University
Liver Dendritic Cell Recruitment and Differentiation During Chronic HCV Infection
Victoria Velazquez Best, PhD, Emory University
Investigating a Protective Role for miR-122 in the Life Cycle of Hepatitis C Virus
Selena Sagan, PhD, Stanford University
Identification of a Novel Host Innate Immune Factor that Regulates HCV
Takeshi Saito, MD, PhD, USC Keck School of Medicine
Development of Novel Host-Factor-Based Antivirals for Hepatitis C
Michael Gelman, MD, PhD, Stanford University
Role of Lipid Droplets in Hepatitis C Virus Infection and Liver Damage
Gregory Camus, PhD, Gladstone Institutes
At least 130 million people worldwide are chronically infected with hepatitis C virus (HCV). Infection frequently leads to advanced liver disease, including fibrosis, cirrhosis and hepatocellular carcinoma. Despite substantial efforts to develop antivirals directly blocking replication, treatment options remain limited.
The current standard of care is only partially effective. A therapeutic or preventative vaccine does not exist. Currently, HCV associated liver transplantation is merely a palliative procedure due to universal infection of the graft after transplantation, often resulting in rapid fibrosis progression and subsequent graft failure.
Even transient therapies inhibiting HCV cell entry could prevent graft reinfection and greatly improve the effectiveness of liver transplantation. Such therapeutic advances targeting this stage of the viral life cycle will require a much more solid understanding of HCV cell entry than is currently available. The development of treatment and prophylactic options has been hampered in part by the lack of immunocompetent, cost effective, robust, and reproducible small animal models for the virus.
Besides humans, chimpanzees are the only species that is naturally susceptible to HCV infection. While experimentation in these large primates has yielded valuable insights, ethical considerations, limited availability, genetic heterogeneity, and cost limit their utility.
To overcome roadblocks to HCV research in vivo, Dr. Ploss’ group has engineered an immunocompetent mouse model with genetically encoded susceptibility to the virus. Generation of HCV-permissive mice was facilitated by his recent discovery that species tropism at the level of entry is defined by two human molecules: CD81 and occludin (OCLN).
Dr. Ploss has demonstrated that transient or stable expression of these entry factors in vivo renders mice susceptible to infection with diverse HCV genotypes. He has shown that viral uptake can be blocked by passive immunization strategies and that immunization of these animals induces humoral immunity and confers partial protection to heterologous challenge. He has also shown proof-of-principle for combining this system with gene knockout analysis to begin to dissect viral entry. He is now proposing to apply this system to genetically dissect HCV infection in vivo.
Dr. Ploss aims to take advantage of existing repositories of mutant mouse strains to assess the impact of targeted gene disruptions on HCV infection. Combining mouse knockout technology with his genetically humanized mouse models therefore offers a unique opportunity dissecting HCV infection in the 3 dimensional context of the liver.
Eighty percent of individuals infected with HCV fail to clear their infection on their own, largely as the result of weak, narrowly targeting or waning antiviral T cell responses. One possibility is that the observed defects in HCV immunity are a direct result of inefficient T cell priming by professional antigen presenting cells (APCs).
Unfortunately, progress in understanding potential defects in antigen presentation during chronic HCV infection has been impeded by the lack of a small animal model in which to study the host response. Using a large cohort of patients undergoing liver transplantation for chronic HCV infection and non HCV-related liver disease, Dr. Best has observed that a number of professional APCs, including dendritic cells (DCs), reside within the human liver microenvironment that have the potential to shape HCV-specific immunity. Among six identified populations of liver APCs, a novel population of CD34+ myeloid ”progenitors” was also discovered.
Interestingly, chronic HCV infection was characterized by a drastic reduction in the frequency of “progenitor” cells and a significant increase in the frequency of intrahepatic myeloid DCs. Moreover, intrahepatic myeloid DCs revealed the HCV-induced expression of numerous maturation markers, including CD80, CD83, CD40 and PD-L1, and were responsive to antigenic stimulation in vitro through the secretion of IL-12.
Using various immunological techniques developed by her laboratory, Dr. Best proposes to test the hypothesis that the chronic HCV liver microenvironment promotes the differentiation of myeloid liver DCs, and that these DCs regulate HCV-specific T cell responses through B7-mediated pathways and the secretion of effector cytokines. Phenotypic analysis of liver “progenitor” cells, and the use of in vitro and in vivo stem cell differentiation assays also allow Dr. Best to determine the recruitment and DC differentiation fate of these cells during chronic HCV infection.
Comparisons of immunomodulatory receptor expression, cytokine secretion potential and antigen presenting capacity of mature liver APCs from uninfected and HCV-infected patients will allow Dr. Best to identify mechanisms of immune system modulation by these cells in the context of chronic HCV infection. Together, the experiments proposed will reveal potential mechanisms underlying HCV chronicity, and will serve useful for the development of novel therapeutic strategies.
Hepatitis C virus (HCV) infection is a rapidly increasing global health problem with over 170 million people infected worldwide. HCV is a hepatotropic, positive-sense RNA virus of the family Flaviviridae. MicroRNA-122 (miR-122) is a highly abundant, liver-specific human microRNA that interacts with two sites in the 5’ end of the HCV RNA genome.
Sequestration of miR-122 leads to a loss of viral RNA in cell culture and in vivo. Curiously, miR-122 does not affect viral translation and has a minimal affect on the rate of RNA synthesis. Dr. Sagan has recently demonstrated that miR-122 binds to the 5’ terminus of the HCV genome, suggesting that miR-122 may protect the terminus of the HCV RNA from attack by nucleases or cellular sensors of RNA.
In this study she will investigate the character of the 5’ terminus of the viral genome during infections. Dr. Sagan will also directly investigate specific gene candidates for their role in HCV RNA recognition and turnover by carrying out short-interfering RNA (siRNA) knockdown during miR-122 sequestration of specific nucleases and cellular sensors of RNA. Finally, a genome-wide siRNA screen will identify genes mediating HCV RNA accumulation during miR-122 sequestration.
The proposed research is highly significant as it examines the mechanism by which miR-122 modulates HCV RNA abundance and will help to identify novel host-virus interactions, defining new targets for therapeutic intervention.
Current antiviral therapy against HCV consists of a combination of pegylated IFN-α (PEG-IFN)
and Ribavirin. However, Dr. Saito recognizes this treatment eradicates HCV in only 40-50% of patients following 48 weeks of treatment and has severe adverse effects. Therefore, improvement of IFN based therapeutic strategy is desperately needed for better therapeutic outcome and relief of medication-related toxicities.
IFN induces antiviral effects through the induction of approximately 200 interferon stimulated genes (ISGs). It is known that HCV proteins block induction and function of some of critical ISGs, leading us to hypothesize that those ISGs are critical to inhibit the HCV life cycle.
In this proposal, Dr. Saito has established a cell line stably expressing the interferon stimulated gene 56 (ISG56) promoter fused to a firefly luciferase reporter gene. Dr. Saito applied the cells to high throughput cDNA screening to identify novel antiviral host factors. The screening identified more than forty genes as potential candidates of antiviral gene inducers including tyrosine kinase, non-receptor, 1(TNK1).
Preliminary experiments suggest that the TNK1 pathway promotes the expression of antiviral genes that suppress HCV replication. Moreover, virus infection and interferon (IFN) treatment each regulate TNK1 abundance, suggesting a regulatory feedback mechanism. These observations indicate that TNK1 is involved in the host response against virus infection and the antiviral action of IFN.
In this proposal, Dr. Saito will focus on defining the mechanism of antiviral gene induction by TNK1 including investigation of the phosphorylation of candidate substrates of TNK1, determination of the signaling cascade of the TNK1 pathway, structure and function analysis of TNK1 to identify its signaling and substrate binding motifs. Furthermore, Dr. Saito will also assess how the antiviral activities of TNK1 control HCV in hepatocytes as a potential novel anti-HCV therapeutic target.
Finally, Dr. Saito will examine the effect of TNK1 combined with pegylated IFN-α (PEG-IFN) and Ribavirin or HCV NS3/4A protease inhibitor as a basis to investigate potential novel anti HCV treatments. Dr. Saito’s study stands to identify novel anti-HCV therapeutic strategies through the identification and characterization of TNK1 mediated antiviral signaling pathways.
The hepatitis C virus (HCV) forms a structure called the “membranous web” in order to replicate. One component of the cellular membranes that are hijacked to form the membranous web is phosphatidylinositol 4,5-bisphosphate (PIP2), a particular membrane lipid molecule. The enzyme that synthesizes PIP2, PI4K IIIα, is required for HCV to replicate. The nonstructural 5A protein (NS5A) of HCV binds specifically to PIP2.
This binding appears to take place at the N-terminal amphipathic helix (AH, an alpha-helix with one charged face and one hydrophobic face) of NS5A. Mutating the AH or sequestering the PIP2 with neomycin disrupts the binding and prevents HCV replication.
Dr. Gelman’s hypothesis is that a novel class of anti-HCV therapeutics that disrupt the PIP2-NS5A interaction can be discovered. One way to disrupt the interaction is to bind either NS5A AH or PIP2. Another approach is to inhibit PI4K IIIα.
Dr. Gelman will pursue both of these approaches. His specific aims are (1) to develop a fluorescence polarization (FP) assay for inhibition of binding of fluorescence-tagged PIP2 to NS5A (AH peptide, full length protein, or biotin-tethered AH peptide), (2) to use this assay to screen thousands of compounds in the Stanford High Throughput Biosciences Center, (3) to validate compounds identified in the screen for activity against HCV replication using luciferase-reporter and focus-reduction assays in cell culture based on the Huh7 or Huh7.5 cell lines, and (4) to screen known and candidate inhibitors of PI4K IIIα to identify compounds that inhibit HCV replication without unacceptable cytotoxicity.
Dr. Gelman expects to identify lead compounds both for disruption of the PIP2-NS5A interaction and for inhibition of PI4K IIIα. Further work will focus on application of organic chemistry expertise to these compounds to synthesize and evaluate promising structural analogues. Since PIP2 is a host factor and PI4K IIIα is a host enzyme, the barrier to development of resistance may be high.
The goal of Dr. Camus’s proposal is to characterize molecular mechanisms controlling Hepatitis C Virus (HCV) replication at lipid droplets (LDs) and leading to liver damages in infected patients. LDs have emerged as central cellular organelles in HCV replication. Triglycerides stored in LDs can be generated by two diacylglycerol acyltransferase (DGAT) enzymes.
Recent results from his laboratory indicate that HCV exclusively replicates at LDs generated by DGAT1. To define the role of DGAT1 in HCV replication, Dr. Camus will 1) characterize proteins interacting specifically with DGAT1 or DGAT2 in hepatoma cells to identify factors that favour or disfavour HCV particle production associated with DGAT1 or DGAT2 respectively. 2) His preliminary results show that two HCV proteins, core and NS5A, interact physically with DGAT1. He will perform a mutagenesis study of DGAT1 and these viral factors to elucidate the role of their interactions in viral assembly. 3) Transgenic mice expressing core develop fatty liver/steatosis, a frequent symptom in HCV-infected individuals. To examine whether DGAT1-generated LDs play a role in core-induced steatosis, he will generate core-transgenic mice in a DGAT1-/- background.
Dr. Camus anticipates that these studies will provide important new insight into the HCV life cycle at LDs and will determine whether DGAT1 is a novel antiviral drug target in HCV infection.