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Berlin Brandenburg

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  • 1
    Language: English
    In: Hepatology (Baltimore, Md.), January 2014, Vol.59(1), pp.24-34
    Description: Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB-1), occludin (OCLN), and claudin-1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh-7.5) or CLDN6 (HuH6). Huh-7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6-dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1-specific neutralizing antibodies (Abs) fully abrogated infection of Huh-7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti-CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1-specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB-1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1-specific therapeutic strategies.
    Keywords: Viral Tropism ; Virus Internalization ; Claudin-1 -- Metabolism ; Claudins -- Metabolism ; Hepacivirus -- Physiology
    E-ISSN: 1527-3350
    Source: MEDLINE/PubMed (U.S. National Library of Medicine)
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  • 2
    In: Hepatology, January 2014, Vol.59(1), pp.24-34
    Description: Hepatitis C Virus (HCV) entry involves at least four cellular factors, including CD81, the scavenger receptor class B type I (SCARB‐1), occludin (OCLN), and claudin‐1 (CLDN1). In addition, CLDN6 and CLDN9 have been shown to substitute for CLDN1 as HCV entry factors in human nonliver cells. We examined the role of different CLDN proteins during HCV entry by using cell lines expressing either predominantly CLDN1 (Huh‐7.5) or CLDN6 (HuH6). Huh‐7.5 cells were susceptible to all tested HCV isolates, whereas HuH6 cells were only permissive to some viral strains. Silencing of CLDN6 in HuH6 cells revealed that these cells are infected in a CLDN6‐dependent fashion, and ectopic expression of CLDN1 or CLDN6 in 293T cells lacking endogenous CLDN expression confirmed that only some HCV strains efficiently use CLDN6 for infection. CLDN1‐specific neutralizing antibodies (Abs) fully abrogated infection of Huh‐7.5 cells by isolates that use CLDN1 only, whereas viruses with broad CLDN tropism were only partially inhibited by these Abs. Importantly, infection by these latter strains in the presence of anti‐CLDN1 Ab was further reduced by silencing CLDN6, suggesting that viruses with broad CLDN usage escape CLDN1‐specific Abs by utilization of CLDN6. Messenger RNA (mRNA) levels of HCV entry factors in liver biopsies of HCV patients infected with different genotype and with variable degree of liver fibrosis were determined. Uniformly high levels of CD81, SCARB‐1, OCLN, and CLDN1 mRNA were detected. In contrast, abundance of CLDN6 mRNA was highly variable between patients. : These findings highlight differential CLDN usage by HCV isolates, which may evolve based on variable expression of CLDN proteins in human liver cells. Broad CLDN tropism may facilitate viral escape from CLDN1‐specific therapeutic strategies. (H 2014;58:24–34)
    Keywords: Antibodies–Immunology ; Biopsy–Metabolism ; Cell Line, Tumor–Metabolism ; Claudin-1–Physiology ; Claudin-1–Metabolism ; Claudins–Pathology ; Hek293 Cells–Metabolism ; Hepacivirus–Metabolism ; Humans–Metabolism ; Liver–Metabolism ; Liver–Metabolism ; RNA, Messenger–Metabolism ; Viral Tropism–Metabolism ; Virus Internalization–Metabolism ; Hepatitis ; Hepatology ; Infections ; Medical Research ; Antibodies ; Cldn1 Protein, Human ; Claudin-1 ; Claudins ; RNA, Messenger ; Claudin 6;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
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  • 3
    Language: English
    In: Hepatology, December 2012, Vol.56(6), pp.2082-2093
    Description: Persistent infection with hepatitis C virus (HCV) can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All current therapies of hepatitis C include interferon‐alpha (IFN‐α). Moreover, IFN‐gamma (IFN‐γ), the only type II IFN, strongly inhibits HCV replication and is the primary mediator of HCV‐specific antiviral T‐cell responses. However, for both cytokines the precise set of effector protein(s) responsible for replication inhibition is not known. The aim of this study was the identification of IFN‐α and IFN‐γ stimulated genes (ISGs) responsible for controlling HCV replication. We devised an RNA interference (RNAi)‐based “gain of function” screen and identified, in addition to known ISGs earlier reported to suppress HCV replication, several new ones with proven antiviral activity. These include IFIT3 (IFN‐induced protein with tetratricopeptide repeats 3), TRIM14 (tripartite motif containing 14), PLSCR1 (phospholipid scramblase 1), and NOS2 (nitric oxide synthase 2, inducible). All ISGs identified in this study were up‐regulated both by IFN‐α and IFN‐γ, demonstrating a substantial overlap of HCV‐specific effectors induced by either cytokine. Nevertheless, some ISGs were more specific for IFN‐α or IFN‐γ, which was most pronounced in case of PLSCR1 and NOS2 that were identified as main effectors of IFN‐γ‐mediated anti‐HCV activity. Combinatorial knockdowns of ISGs suggest additive or synergistic effects demonstrating that with either IFN, inhibition of HCV replication is caused by the combined action of multiple ISGs. Conclusion: Our study identifies a number of novel ISGs contributing to the suppression of HCV replication by type I and type II IFN. We demonstrate a substantial overlap of antiviral programs triggered by either cytokine and show that suppression of HCV replication is mediated by the concerted action of multiple effectors. (H 2012;56:2082–2093)
    Keywords: Antigens, Differentiation–Genetics ; Antigens, Differentiation–Metabolism ; Carrier Proteins–Genetics ; Carrier Proteins–Metabolism ; Gene Expression Regulation–Physiology ; Hepacivirus–Drug Effects ; Hepatocytes–Metabolism ; Hepatocytes–Pharmacology ; Humans–Pharmacology ; Interferon-Alpha–Genetics ; Interferon-Gamma–Metabolism ; Intracellular Signaling Peptides & Proteins–Genetics ; Intracellular Signaling Peptides & Proteins–Metabolism ; Membrane Proteins–Genetics ; Membrane Proteins–Metabolism ; Nitric Oxide Synthase Type II–Genetics ; Nitric Oxide Synthase Type II–Metabolism ; Phospholipid Transfer Proteins–Metabolism ; Phospholipid Transfer Proteins–Genetics ; RNA Interference–Metabolism ; RNA, Messenger–Drug Effects ; RNA-Binding Proteins–Drug Effects ; RNA-Binding Proteins–Drug Effects ; Replicon–Drug Effects ; Tumor Cells, Cultured–Drug Effects ; Up-Regulation–Drug Effects ; Virus Replication–Drug Effects ; Liver Cirrhosis ; Hepatitis ; Cytokines ; Hepatology ; Antigens, Differentiation ; Carrier Proteins ; Ifit3 Protein, Human ; Ifitm3 Protein, Human ; Interferon-Alpha ; Intracellular Signaling Peptides and Proteins ; Membrane Proteins ; Plscr1 Protein, Human ; Phospholipid Transfer Proteins ; RNA, Messenger ; RNA-Binding Proteins ; Trim14 Protein, Human ; Leu-13 Antigen ; Interferon-Gamma ; Nos2 Protein, Human ; Nitric Oxide Synthase Type II;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
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  • 4
    In: Hepatology, August 2014, Vol.60(2), pp.508-520
    Description: The Japanese fulminant hepatitis‐1 (JFH1)‐based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle . However, lack of robust infection systems for primary, patient‐derived isolates limits systematic functional studies of viral intrahost variation and vaccine development. Therefore, we aimed at developing cell culture models for incorporation of primary patient‐derived glycoproteins into infectious HCV particles for in‐depth mechanistic studies of envelope gene function. To this end, we first constructed a packaging cell line expressing core, p7, and NS2 based on the highly infectious Jc1 genotype (GT) 2a chimeric genome. We show that this packaging cell line can be transfected with HCV replicons encoding cognate Jc1‐derived glycoprotein genes for production of single‐round infectious particles by way of ‐complementation. Testing replicons expressing representative envelope protein genes from all major HCV genotypes, we observed that virus production occurred in a genotype‐ and isolate‐dependent fashion. Importantly, primary GT 2 patient‐derived glycoproteins were efficiently incorporated into infectious particles. Moreover, replacement of J6 (GT 2a) core, p7, and NS2 with GT 1a‐derived H77 proteins allowed production of infectious HCV particles with GT 1 patient‐derived glycoproteins. Notably, adaptive mutations known to enhance virus production from GT 1a‐2a chimeric genomes further increased virus release. Finally, virus particles with primary patient‐derived E1‐E2 proteins possessed biophysical properties comparable to Jc1 HCVcc particles, used CD81 for cell entry, were associated with ApoE and could be neutralized by immune sera. : This work describes cell culture systems for production of infectious HCV particles with primary envelope protein genes from GT 1 and GT 2‐infected patients, thus opening up new opportunities to dissect envelope gene function in an individualized fashion. (H 2014;60:508–520)
    Keywords: Antibodies, Monoclonal–Immunology ; Antigens, Cd81–Metabolism ; Apolipoproteins E–Metabolism ; Genetic Complementation Test–Metabolism ; Glycoproteins–Immunology ; Hek293 Cells–Metabolism ; Hepacivirus–Immunology ; Hepacivirus–Metabolism ; Hepatitis C–Virology ; Hepatitis C–Genetics ; Hepatitis C–Metabolism ; Humans–Genetics ; Neutralization Tests–Metabolism ; Phylogeny–Immunology ; RNA, Viral–Immunology ; RNA, Viral–Metabolism ; Viral Envelope Proteins–Immunology ; Viral Envelope Proteins–Physiology ; Viral Hepatitis Vaccines–Physiology ; Virion–Physiology ; Virion–Physiology ; Virus Replication–Physiology ; Virus Replication–Physiology ; Hepatitis ; Atoms & Subatomic Particles ; Hepatology ; Antibodies, Monoclonal ; Antigens, Cd81 ; Apolipoproteins E ; Cd81 Protein, Human ; E1 Protein, Hepatitis C Virus ; Glycoproteins ; RNA, Viral ; Viral Envelope Proteins ; Viral Hepatitis Vaccines ; Glycoprotein E2, Hepatitis C Virus;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
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  • 5
    In: Hepatology, January 2016, Vol.63(1), pp.49-62
    Description: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry and in a genotype‐dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine‐resistant HCV carried mutations within the alleged fusion peptide and displayed cross‐resistance to these compounds, indicating that these drugs have a common mode of action. : These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first‐in‐class HCV fusion inhibitors that merit consideration for repurposing as a cost‐effective component of HCV combination therapies. (H 2016;63:49–62)
    Keywords: Replication ; Membrane Fusion ; Migraine ; Headache ; Phenothiazine ; Glycoproteins ; Hepatitis C ; Genotypes ; Infection ; Mutation ; Cross-Resistance ; Drugs ; Hepatitis C Virus ; Replication ; Microorganisms & Parasites;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
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  • 6
    In: Hepatology, January 2014, Vol.59(1), pp.78-88
    Description: Only humans and chimpanzees are susceptible to chronic infection by hepatitis C virus (HCV). The restricted species tropism of HCV is determined by distinct host factor requirements at different steps of the viral life cycle. In addition, effective innate immune targeting precludes efficient propagation of HCV in nonhuman cells. Species‐specificity of HCV host factor usage for cell entry and virus release has been explored. However, the reason for inefficient HCV RNA replication efficiency in mouse liver cells remains elusive. To address this, we generated novel mouse liver‐derived cell lines with specific lesions in mitochondrial antiviral signaling protein (MAVS), interferon regulatory factor 3 (IRF3), or Interferon‐α/β receptor (IFNAR) by immortalization. Blunted innate immune responses in these cells modestly increased HCV RNA replication. However, ectopic expression of liver‐specific human microRNA 122 (miR‐122) further boosted RNA replication in all knockout cell lines. Remarkably, MAVSmiR‐122 cells sustained vigorous HCV RNA replication, attaining levels comparable to the highly permissive human hepatoma cell line Huh‐7.5. RNA replication was dependent on mouse cyclophilin and phosphatidylinositol‐4 kinase III alpha (PI4KIIIα) and was also observed after transfection of full‐length viral RNA. Additionally, ectopic expression of either human or mouse apolipoprotein E (ApoE) was sufficient to permit release of infectious particles. Finally, expression of human entry cofactors rendered these cells permissive to HCV infection, thus confirming that all steps of the HCV replication cycle can be reconstituted in mouse liver‐derived cells. : Blunted innate immunity, abundant miR‐122, and HCV entry factor expression permits propagation of HCV in mouse liver‐derived cell lines. (H 2014;58:78–88)
    Keywords: 1-Phosphatidylinositol 4-Kinase–Metabolism ; Adaptor Proteins, Signal Transducing–Metabolism ; Animals–Metabolism ; Apolipoproteins E–Metabolism ; Cell Line, Tumor–Physiology ; Cyclophilins–Virology ; Hepacivirus–Metabolism ; Humans–Metabolism ; Immunity, Innate–Metabolism ; Liver–Metabolism ; Mice–Metabolism ; Mice, Knockout–Metabolism ; Micrornas–Metabolism ; RNA, Viral–Metabolism ; Virus Internalization–Metabolism ; Virus Replication–Metabolism ; Hepatitis ; Liver ; Rodents ; Kinases ; Adaptor Proteins, Signal Transducing ; Apolipoproteins E ; IPS-1 Protein, Mouse ; Micrornas ; Mirn122 Microrna, Mouse ; RNA, Viral ; 1-Phosphatidylinositol 4-Kinase ; Cyclophilins;
    ISSN: 0270-9139
    E-ISSN: 1527-3350
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