Transient Replication of Hepatitis C Virus Sub-Genomic RNA in Murine Cell Lines Is Enabled by miR-122 and Varies with Cell Passage

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of viral entry and infection has been hampered by the inability to efficiently propagate the virus in cultured cells and the lack of a small-animal model. Recent studies have shown that in insect cells, the HCV structural proteins assemble into HCV-like particles (HCV-LPs) with morphological, biophysical, and antigenic properties similar to those of putative virions isolated from HCV-infected humans. In this study, we used HCV-LPs derived from infectious clone H77C as a tool to examine virus-cell interactions. The binding of partially purified particles to human cell lines was analyzed by fluorescence-activated cell sorting with defined monoclonal antibodies to envelope glycoprotein E2. HCV-LPs demonstrated dose-dependent and saturable binding to defined human lymphoma and hepatoma cell lines but not to mouse cell lines. Binding could be inhibited by monoclonal anti-E2 antibodies, indicating that the HCV-LP-cell interaction was mediated by envelope glycoprotein E2. Binding appeared to be CD81 independent and did not correlate with low-density lipoprotein receptor expression. Heat denaturation of HCV-LPs drastically reduced binding, indicating that the interaction of HCV-LPs with target cells was dependent on the proper conformation of the particles. In conclusion, our data demonstrate that insect cell-derived HCV-LPs bind specifically to defined human cell lines. Since the envelope proteins of HCV-LPs are presumably presented in a virion-like conformation, the binding of HCV-LPs to target cells may allow the study of virus-host cell interactions, including the isolation of HCV receptor candidates and antibody-mediated neutralization of binding.

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Examination of the Activity of Camel Milk Casein against Hepatitis C Virus (Genotype-4a) and Its Apoptotic Potential in Hepatoma and HeLa Cell Lines

Hepatitis Monthly ◽

10.5812/kowsar.1735143x.722 ◽

2011 ◽

Vol 11 (9) ◽

Cited By ~ 4

Author(s):

Osama Almahdy ◽

Esmail M. EL-Fakharany ◽

Ehab EL-Dabaa ◽

Tzi Bun Ng ◽

Elrashdy M. Redwan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hela Cell ◽

Cell Lines ◽

Camel Milk ◽

Virus Genotype ◽

Hela Cell Lines ◽

Milk Casein

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Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells

Journal of Virology ◽

10.1128/jvi.79.22.13963-13973.2005 ◽

2005 ◽

Vol 79 (22) ◽

pp. 13963-13973 ◽

Cited By ~ 120

Author(s):

Zhaohui Cai ◽

Chen Zhang ◽

Kyung-Soo Chang ◽

Jieyun Jiang ◽

Byung-Chul Ahn ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Culture System ◽

Infectious Virus ◽

Hcv Infection ◽

Hcv Rna ◽

Human Hepatoma ◽

Stable Cell Lines ◽

Increased Risk

ABSTRACT Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5′ end and a hepatitis delta virus ribozyme at the 3′ end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 108 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

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Patient-derived hepatitis C virus and JFH-1 clones differ in their ability to infect human hepatoma cells and lymphocytes

Journal of General Virology ◽

10.1099/vir.0.045393-0 ◽

2012 ◽

Vol 93 (11) ◽

pp. 2399-2407 ◽

Cited By ~ 8

Author(s):

Mohammed A. Sarhan ◽

Annie Y. Chen ◽

Rodney S. Russell ◽

Tomasz I. Michalak

Keyword(s):

T Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

T Cell ◽

Cell Lines ◽

Productive Infection ◽

Wild Type Virus ◽

Human Hepatoma ◽

Naturally Occurring ◽

T Cell Lines

Hepatitis C virus (HCV) is a hepatotropic virus that also infects cells of the immune system. HCV clones cultivated in human hepatoma Huh-7.5 cells have significantly advanced our understanding of HCV replication and candidate hepatocyte receptors. However, naturally occurring patient-derived HCV, in contrast to the HCV JFH-1 clone, is unable to infect Huh-7.5 cells, while it can replicate in human primary T-cells and selected T-cell lines. To better understand this incongruity, we examined the susceptibility of primary T-cells, PBMCs and T-cell lines to infection with patient-derived HCV, the classical HCV JFH-1 and a cell culture-adapted JFH1T known to be highly infectious to Huh-7.5 cells. We also tested whether Huh-7.5 cells are prone to virus readily infecting T-lymphocytes. The results revealed that while primary T-cells and Molt4 and Jurkat T-cell lines were susceptible to patient-derived HCV, they were resistant to infection with either JFH1T or JFH-1. However, the JFH1T clone interacted more firmly, although non-productively, with the cells than JFH-1. Further, Huh-7.5 cells robustly supported replication of JFH1T but not patient-derived, wild-type virus, despite using highly sensitive detection assays. In conclusion, JFH-1 and JFH1T clones were unable to establish productive infection in human primary T-cells, PBMCs and T-cell lines known to be prone to infection by patient-derived HCV, while Huh-7.5 cells were resistant to infection with naturally occurring virus infecting immune cells. The data showed that the ability to infect lymphocytes is a characteristic of native virus but not laboratory HCV clones.

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Hepatitis C virus proteins interfere with the activation of chemokine gene promoters and downregulate chemokine gene expression

Journal of General Virology ◽

10.1099/vir.0.83316-0 ◽

2008 ◽

Vol 89 (2) ◽

pp. 432-443 ◽

Cited By ~ 16

Author(s):

Maarit Sillanpää ◽

Pasi Kaukinen ◽

Krister Melén ◽

Ilkka Julkunen

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Protein Complex ◽

Sendai Virus ◽

Adaptor Protein ◽

Mitogen Activated Protein Kinase ◽

Osteosarcoma Cell ◽

Gene Promoters ◽

Chemokine Gene

The hepatitis C virus (HCV) non-structural (NS) 3/4A protein complex inhibits the retinoic acid inducible gene I (RIG-I) pathway by proteolytically cleaving mitochondria-associated CARD-containing adaptor protein Cardif, and this leads to reduced production of beta interferon (IFN-β). This study examined the expression of CCL5 (regulated upon activation, normal T-cell expressed and secreted, or RANTES), CXCL8 (interleukin 8) and CXCL10 (IFN-γ-activated protein 10, or IP-10) chemokine genes in osteosarcoma cell lines that inducibly expressed NS3/4A, NS4B, core-E1-E2-p7 and the entire HCV polyprotein. Sendai virus (SeV)-induced production of IFN-β, CCL5, CXCL8 and CXCL10 was downregulated by the NS3/4A protein complex and by the full-length HCV polyprotein. Expression of NS3/4A and the HCV polyprotein reduced the binding of interferon regulatory factors (IRFs) 1 and 3 and, to a lesser extent, nuclear factor (NF)-κB (p65/p50) to their respective binding elements on the CXCL10 promoter during SeV infection. Furthermore, binding of IRF1 and IRF3 to the interferon-stimulated response element-like element, and of c-Jun and phosphorylated c-Jun to the activator protein 1 element of the CXCL8 promoter, was reduced when NS3/4A and the HCV polyprotein were expressed. In cell lines expressing NS3/4A and the HCV polyprotein, the subcellular localization of mitochondria was changed, and this was kinetically associated with the partial degradation of endogenous Cardif. These results indicate that NS3/4A alone or as part of the HCV polyprotein disturbs the expression of IRF1- and IRF3-regulated genes, as well as affecting mitogen-activated protein kinase kinase- and NF-κB-regulated genes.

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Poly(I:C) and Lipopolysaccharide Innate Sensing Functions of Circulating Human Myeloid Dendritic Cells Are Affected In Vivo in Hepatitis C Virus-Infected Patients

Journal of Virology ◽

10.1128/jvi.01741-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5537-5546 ◽

Cited By ~ 31

Author(s):

Ian Gaël Rodrigue-Gervais ◽

Loubna Jouan ◽

Geneviève Beaulé ◽

Dominike Sauvé ◽

Julie Bruneau ◽

...

Keyword(s):

Dendritic Cells ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Poly I:C ◽

Hcv Rna ◽

Genomic Rna ◽

Tnf Α ◽

Wide Range ◽

Rna Levels

ABSTRACT The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log10, 5.0 per 106 cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-α− IL-12− cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs (∼6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena.

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