Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells

Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well-characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV-infected cells. V protein-transduced HFLC supported enhanced (10 to 100-fold) levels of HCV infection relative to untransduced or control vector-transduced HFLC. Infection was assessed by measurement of virus-driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV-infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein-transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV-inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein-transduced cultures. Conclusion: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population.National Institutes of Health (U.S.) (NIH Roadmap for Medical Research Grant 1 R01 DK085713-01)Greenberg Institute for Medical ResearchStarr Foundatio

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

A genetically humanized mouse model for hepatitis C virus infection

Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo

Scavenger receptor class B type 1 is an essential receptor for Hepatitis C virus and a potential target for novel therapies

L’epatite C costituisce un problema sanitario di importanza globale e si stima che circa il 3% della popolazione mondiale sviluppi epatite C cronica (circa 170 milioni di persone). Un vaccino per l’epatite C non e’ ancora disponibile e le attuali terapie hanno un’efficacia limitata, costi elevati e effetti collaterali notevoli. Vi e’ pertanto un urgente bisogno di sviluppare nuove terapie che agiscano attraverso differenti meccanismi d’azione. Recenti scoperte nel campo della biologia di HCV hanno portato ad una migliore comprensione dei meccanismi molecolari di legame e ingresso del virus nelle cellule bersaglio con possibili ripercussioni sul disegno di strategie innovative per lo sviluppo di nuove molecole ad azione profilattica e terapeutica. In questo lavoro mi sono interessata dei meccanismi di interazione del virus con la membrana delle cellule bersaglio, che costituiscono il primo evento nel processo di infezione. In particolare, ho studiato il ruolo di un nuovo possibile recettore per HCV, lo scavenger receptor class B type 1 (SR-B1), nei processi di entry e spreading. A tal fine, ho sviluppato diversi saggi per misurare sia il legame del virus alle cellule epatiche che l’infezione. Ho inoltre generato reagenti, come ad esempio anticorpi monoclonali e siRNAs per caratterizzare il ruolo svolto da SR-B1 nel processo di infezione da HCV. Per valutare in quale fase dell’infezione fosse coinvolto SR-B1 sono state utilizzate particelle virali prodotte sia in vitro che in vivo. Questo progetto di ricerca ha permesso di dimostrare che SR-B1 costituisce un fattore essenziale per l’infezione da parte di HCV e che e’ coinvolto nelle fasi piu’ precoci del processo di riconoscimento virus/cellula. A causa del suo ruolo fisiologico nel metabolismo delle lipoproteine, ho anche analizzato l’importanza delle lipoproteine nell’infezione da HCV e ho potuto dimostrare che le lipoproteine ad alta densita’ (HDL) sono in grado di potenziare l’infezione in cellule in coltura. Inoltre, mediante mutagenesi sito-specifica, ho potuto identificare residui aminoacidici di SR-B1 coinvolti nel riconoscimento della glicoproteina di HCV, E2 e ho dimostrato che questi residui svolgono un ruolo importante anche nel legame della particella virale ad SR-B1. In conclusione, tale lavoro dimostra il ruolo essenziale di SR-B1 nell’ingresso di HCV nelle cellule bersaglio e allo stesso tempo getta le basi per approcci terapeutici alternativi che abbiano come bersaglio molecole esposte sulle cellule dell’ospite piuttosto che sulla superficie virale, consentendo cosi’ di superare i problemi di inefficacia dei trattamenti consueti dovuti alla rapida insorgenza di varianti virali.Hepatitis C virus (HCV) is an emerging infection and a worldwide medical problem with 3% of the world's population being chronically infected (approximately 170 million people). A vaccine against HCV is not available and current antiviral therapies have limited efficacy, high costs and significant side effects. There is a clear and pressing need for the development of additional therapies that act through different mechanisms. Recent discoveries in the HCV biology field have led to a better knowledge of the molecular mechanisms of HCV binding and entry into target cells with potential impact on novel strategies for the development of new preventative and therapeutic agents. In this work, I investigated the mechanism of virus interaction with the cell surface of target cells to initiate the infection process. More specifically, I addressed the role of a novel HCV receptor candidate, the scavenger receptor class B type 1 (SR-B1), in HCV entry and spread. To this end, I set up a number of assays for measuring in vitro binding and infection and have generated reagents, including antibodies and small interfering RNAs (siRNAs), to characterize the process of SR-B1 mediated HCV infection. In vitro and in vivo-produced HCV viral particles were used to dissect the molecular steps of virus entry through SR-B1. This work led to the conclusion that SR-B1 is an essential factor for HCV entry and is involved in an early event of virus/cell recognition. Because of its physiological role in lipoprotein metabolism, I also investigated the importance of lipoproteins in HCV infection and could show that high density lipoprotein (HDL) can enhance HCV infection in cell culture. Finally, by site-specific mutagenesis, I could identify critical residues within SR-B1 protein sequence involved in the recognition of the HCV glycoprotein E2 and have demonstrated that these residues play a role in receptor recognition by the real virus. While highlighting the essential role of SR-B1 in HCV entry, this work provides insights into alternative approaches for counteracting HCV infection by exploiting cellular rather than viral targets, thus overcoming the problem of viral variability

Scavenger receptor class B type 1 is an essential receptor for Hepatitis C virus and a potential target for novel therapies

L’epatite C costituisce un problema sanitario di importanza globale e si stima che circa il 3% della popolazione mondiale sviluppi epatite C cronica (circa 170 milioni di persone). Un vaccino per l’epatite C non e’ ancora disponibile e le attuali terapie hanno un’efficacia limitata, costi elevati e effetti collaterali notevoli. Vi e’ pertanto un urgente bisogno di sviluppare nuove terapie che agiscano attraverso differenti meccanismi d’azione. Recenti scoperte nel campo della biologia di HCV hanno portato ad una migliore comprensione dei meccanismi molecolari di legame e ingresso del virus nelle cellule bersaglio con possibili ripercussioni sul disegno di strategie innovative per lo sviluppo di nuove molecole ad azione profilattica e terapeutica. In questo lavoro mi sono interessata dei meccanismi di interazione del virus con la membrana delle cellule bersaglio, che costituiscono il primo evento nel processo di infezione. In particolare, ho studiato il ruolo di un nuovo possibile recettore per HCV, lo scavenger receptor class B type 1 (SR-B1), nei processi di entry e spreading. A tal fine, ho sviluppato diversi saggi per misurare sia il legame del virus alle cellule epatiche che l’infezione. Ho inoltre generato reagenti, come ad esempio anticorpi monoclonali e siRNAs per caratterizzare il ruolo svolto da SR-B1 nel processo di infezione da HCV. Per valutare in quale fase dell’infezione fosse coinvolto SR-B1 sono state utilizzate particelle virali prodotte sia in vitro che in vivo. Questo progetto di ricerca ha permesso di dimostrare che SR-B1 costituisce un fattore essenziale per l’infezione da parte di HCV e che e’ coinvolto nelle fasi piu’ precoci del processo di riconoscimento virus/cellula. A causa del suo ruolo fisiologico nel metabolismo delle lipoproteine, ho anche analizzato l’importanza delle lipoproteine nell’infezione da HCV e ho potuto dimostrare che le lipoproteine ad alta densita’ (HDL) sono in grado di potenziare l’infezione in cellule in coltura. Inoltre, mediante mutagenesi sito-specifica, ho potuto identificare residui aminoacidici di SR-B1 coinvolti nel riconoscimento della glicoproteina di HCV, E2 e ho dimostrato che questi residui svolgono un ruolo importante anche nel legame della particella virale ad SR-B1. In conclusione, tale lavoro dimostra il ruolo essenziale di SR-B1 nell’ingresso di HCV nelle cellule bersaglio e allo stesso tempo getta le basi per approcci terapeutici alternativi che abbiano come bersaglio molecole esposte sulle cellule dell’ospite piuttosto che sulla superficie virale, consentendo cosi’ di superare i problemi di inefficacia dei trattamenti consueti dovuti alla rapida insorgenza di varianti virali.Hepatitis C virus (HCV) is an emerging infection and a worldwide medical problem with 3% of the world's population being chronically infected (approximately 170 million people). A vaccine against HCV is not available and current antiviral therapies have limited efficacy, high costs and significant side effects. There is a clear and pressing need for the development of additional therapies that act through different mechanisms. Recent discoveries in the HCV biology field have led to a better knowledge of the molecular mechanisms of HCV binding and entry into target cells with potential impact on novel strategies for the development of new preventative and therapeutic agents. In this work, I investigated the mechanism of virus interaction with the cell surface of target cells to initiate the infection process. More specifically, I addressed the role of a novel HCV receptor candidate, the scavenger receptor class B type 1 (SR-B1), in HCV entry and spread. To this end, I set up a number of assays for measuring in vitro binding and infection and have generated reagents, including antibodies and small interfering RNAs (siRNAs), to characterize the process of SR-B1 mediated HCV infection. In vitro and in vivo-produced HCV viral particles were used to dissect the molecular steps of virus entry through SR-B1. This work led to the conclusion that SR-B1 is an essential factor for HCV entry and is involved in an early event of virus/cell recognition. Because of its physiological role in lipoprotein metabolism, I also investigated the importance of lipoproteins in HCV infection and could show that high density lipoprotein (HDL) can enhance HCV infection in cell culture. Finally, by site-specific mutagenesis, I could identify critical residues within SR-B1 protein sequence involved in the recognition of the HCV glycoprotein E2 and have demonstrated that these residues play a role in receptor recognition by the real virus. While highlighting the essential role of SR-B1 in HCV entry, this work provides insights into alternative approaches for counteracting HCV infection by exploiting cellular rather than viral targets, thus overcoming the problem of viral variability

Advances in experimental systems to study hepatitis C virus in vitro and in vivo

AbstractHepatitis C virus (HCV) represents a global health concern affecting over 185 million people worldwide. Chronic HCV infection causes liver fibrosis and cirrhosis and is the leading indication for liver transplantation. Recent advances in the field of direct-acting antiviral drugs (DAAs) promise a cure for HCV in over 90% of cases that will get access to these expensive treatments. Nevertheless, the lack of a protective vaccine and likely emergence of drug-resistant viral variants call for further studies of HCV biology. With chimpanzees being for a long time the only non-human in vivo model of HCV infection, strong efforts were put into establishing in vitro experimental systems. The initial models only enabled to study specific aspects of the HCV life cycle, such as viral replication with the subgenomic replicon and entry using HCV pseudotyped particles (HCVpp). Subsequent development of protocols to grow infectious HCV particles in cell-culture (HCVcc) ignited investigations on the full cycle of HCV infection and the virus–host interactions required for virus propagation.More recently, small animal models permissive to HCV were generated that allowed in vivo testing of novel antiviral therapies as well as vaccine candidates. This review provides an overview of the currently available in vitro and in vivo experimental systems to study HCV biology. Particular emphasis is given to how these model systems furthered our understanding of virus–host interactions, viral pathogenesis and immunological responses to HCV infection, as well as drug and vaccine development

The interferon inducible isoform of NCOA7 inhibits endosome-mediated viral entry

International audienceInterferons (IFNs) mediate cellular defence against viral pathogens by upregulation of IFN-stimulated genes whose products interact with viral components or alter cellular physiology to suppress viral replication1-3. Among the IFN-stimulated genes that can inhibit influenza A virus (IAV)4 are the myxovirus resistance 1 GTPase5 and IFN-induced transmembrane protein 3 (refs 6,7). Here, we use ectopic expression and gene knockout to demonstrate that the IFN-inducible 219-amino acid short isoform of human nuclear receptor coactivator 7 (NCOA7) is an inhibitor of IAV as well as other viruses that enter the cell by endocytosis, including hepatitis C virus. NCOA7 interacts with the vacuolar H+-ATPase (V-ATPase) and its expression promotes cytoplasmic vesicle acidification, lysosomal protease activity and the degradation of endocytosed antigen. Step-wise dissection of the IAV entry pathway demonstrates that NCOA7 inhibits fusion of the viral and endosomal membranes and subsequent nuclear translocation of viral ribonucleoproteins. Therefore, NCOA7 provides a mechanism for immune regulation of endolysosomal physiology that not only suppresses viral entry into the cytosol from this compartment but may also regulate other V-ATPase-associated cellular processes, such as physiological adjustments to nutritional status, or the maturation and function of antigen-presenting cells

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus–host interactions underlying HCV infection