Targeting HCV Entry For Development of Therapeutics

Recent progress in defining the molecular mechanisms of Hepatitis C Virus (HCV) entry affords the opportunity to exploit new viral and host targets for therapeutic intervention. Entry inhibitors would limit the expansion of the infected cell reservoir, and would complement the many replication inhibitors now under development. The current model for the pathway of entry involves the initial docking of the virus onto the cell surface through interactions of virion envelope and associated low density lipoproteins (LDL) with cell surface glycosaminoglycans and lipoprotein receptors, followed by more specific utilization with other hepatocyte membrane proteins: Scavenger Receptor Class B type 1 (SR-BI), CD81, Claudin 1 (CLDN1) and Occludin (OCLN). The use of blockers of these interactions, e.g. specific antibodies, suggests that inhibition of any one step in the entry pathway can inhibit infection. Despite this knowledge base, the tools for compound screening, HCV pseudoparticles (HCVpp) and cell culture virus (HCVcc), and the ability to adapt them to industrial use are only recently available and as a result drug discovery initiatives are in their infancy. Several therapies aiming at modulating the virus envelope to prevent host cell binding are in early clinical testing. The first test case for blocking a cellular co-receptor is an SR-BI modulator. ITX 5061, an orally active small molecule, targets SR-BI and has shown potent antiviral activity against HCVpp and HCVcc. ITX 5061 has exhibited good safety in previous clinical studies, and is being evaluated in the clinic in chronic HCV patients and patients undergoing liver transplantation. Entry inhibitors promise to be valuable players in the future development of curative therapy against HCV

Complete replication of hepatitis C virus in cell culture.

Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10(5) infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-alpha and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals

Neutralizing Antibody-Resistant Hepatitis C Virus Cell-to-Cell Transmission

Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal anti- bodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins rep- resenting the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell trans- mission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver

Genetic Mosaic Analysis Indicates That the Bulb Region of Coat Hair Follicles Contains a Resident Population of Several Active Multipotent Epithelial Lineage Progenitors

AbstractThe hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal–distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia

Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system

Author Manuscript 2010 August 1Hepatitis C virus (HCV), which infects 2–3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation1. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.New York (State). Dept. of Health (Empire State Stem Cell Fund Contract C023046)United States. Public Health Service (Grant R01 DK56966)National Institutes of Health (U.S.) (Roadmap for Medical Research Grant 1 R01 DK085713-01)Howard Hughes Medical Institute (Investigator

Genomic Analysis Reveals a Potential Role for Cell Cycle Perturbation in HCV-Mediated Apoptosis of Cultured Hepatocytes

The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death–related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-β1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation

Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics

Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis

A molecular profile of the mouse gastric parietal cell with and without exposure to Helicobacter pylori

The parietal cell (PC) plays an important role in normal gastric physiology and in common diseases of the stomach. Although the genes involved in acid secretion are well known, there is limited molecular information about other aspects of PC function. We have generated a comprehensive database of genes expressed preferentially in PCs relative to other gastric mucosal cell lineages. PCs were purified from FVB/N mouse stomachs by lectin panning. cRNA generated from PC-enriched (PC(+)) and PC-depleted (PC(−)) populations were used to query oligonucleotide-based microarrays. False-positive signals were filtered by using a new algorithm for noise reduction and selected results independently audited by real-time quantitative reverse transcription (RT)-PCR. The annotated database of 240 genes reveals previously unappreciated aspects of cellular function, including factors that may mediate PC regulation of gastric stem cell proliferation. PC(+) and PC(−) expression profiles were also prepared from germ-free mice 2 and 8 weeks after colonization with a clinical isolate of Helicobacter pylori (Hp)—the pathogen that produces acid-peptic disease (gastritis, ulcers) in humans. Whereas PC(+) gene expression was remarkably constant, the PC(−) fractions demonstrated a robust, evolving host response, with increased expression of genes involved in cell motility/migration, extracellular matrix interactions, and IFN responses. The consistency of PC(+) gene expression allowed identification of a cohort of 92 genes enriched in PCs under all conditions studied. These genes provide a molecular profile that can be used to define this epithelial lineage under a variety of physiologic, pharmacologic, and pathologic stimuli