Martina Friesland

Hannover Medical School, Hanover, Lower Saxony, Germany

Are you Martina Friesland?

Claim your profile

Publications (26)121.11 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) infection is a major biomedical problem worldwide as it causes severe liver disease in millions of humans around the world. Despite the recent approval of specific drugs targeting HCV replication to be used in combination with interferon-α and ribavirin, there is still an urgent need for pangenotypic, interferon-free therapies to fight this genetically diverse group of viruses. In this study, we have used an unbiased screening cell culture assay to interrogate a chemical library of compounds approved for clinical use in humans. This system enables identifying non-toxic antiviral compounds targeting every aspect of the viral lifecycle, be the target viral or cellular. The aim of this study is to identify drugs approved for other therapeutic applications in humans that could be effective components of combination therapies against HCV. As a result of this analysis we identified twelve compounds with antiviral activity in cell culture, some of which had previously been identified as HCV inhibitors with antiviral activity in cell culture and shown to be effective in patients. We selected two novel HCV antivirals, hydroxyzine and benztropin, to characterize them by determining their specificity and genotype spectrum as well as by defining the step of the replication cycle targeted by these compounds. We found that both compounds effectively inhibited viral entry at a post-binding step of genotypes 1, 2, 3 and 4 without affecting entry of other viruses.
    Antimicrobial Agents and Chemotherapy 04/2014; · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The JFH1-based hepatitis C virus (HCV) infection system has permitted analysis of the complete viral replication cycle in vitro. However, lack of robust infection systems for primary, patient-derived isolates limits systematic functional studies of viral intra-host 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 trans-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. Conclusion: 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. (Hepatology 2014)
    Hepatology 04/2014; · 12.00 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) is highly variable and associated with chronic liver disease. Viral isolates are grouped into seven genotypes (GTs). Accumulating evidence indicates that viral determinants in the core to NS2 proteins modulate the efficiency of virus production. However, the role of the glycoproteins E1 and E2 in this process is currently poorly defined. Therefore, we constructed chimeric viral genomes to explore the role of E1 and E2 in HCV assembly. Comparison of the kinetics and efficiency of particle production by intra-genotypic chimeras highlighted core and p7 as crucial determinants for efficient virion release. Glycoprotein sequences, however, had only minimal impact on this process. In contrast, in the context of inter-genotypic HCV chimeras, HCV assembly was profoundly influenced by glycoprotein genes. On the one hand, insertion of GT 1a-derived (H77) E1-E2 sequences into a chimeric GT2a virus (Jc1) strongly suppressed virus production. On the other hand, replacement of H77 glycoproteins within the GT1a-2a chimeric genome H77/C3 by GT2a-derived (Jc1) E1-E2 increased infectious particle production. Thus, within inter-genotypic chimeras glycoprotein features strongly modulate virus production. Replacement of Jc1 glycoprotein genes by H77-derived E1-E2 did not grossly affect subcellular localization of core, E2 and NS2. However, it caused an accumulation of non-enveloped core protein, and increased abundance of non-enveloped core protein structures with slow sedimentation. These findings reveal an important role for the HCV glycoproteins E1 and E2 in membrane envelopment which likely depends on a genotype-specific interplay with additional viral factors.
    Journal of Virology 10/2013; · 5.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background. Hepatitis C virus (HCV) is spread through direct contact with blood, although alternative routes of transmission may contribute to the global burden. Perinatal infection occurs in up to 5 % of HCV infected mothers and presence of HCV RNA in breast milk has been reported. We investigated the influence of breast milk on HCV infectiousness.Methods/Results. Human breast milk reduced HCV infectivity in a dose-dependent manner. This effect was species-specific since milk from various animals did not inhibit HCV infection. Treatment of HCV with human breast milk did not compromise integrity of viral RNA or capsids, but destroyed the lipid envelope. Fractionation of breast milk revealed that the antiviral activity is present in the cream fraction containing the fat. Proteolytic digestion of milk proteins had no influence on its antiviral activity whereas prolonged storage at 4 °C increased antiviral activity. Notably, pretreatment with a lipase inhibitor ablated the antiviral activity and specific free fatty acids of breast milk were antiviral.Conclusion. The antiviral activity of breast milk is linked to endogenous lipase-dependent generation of free fatty acids which destroy the viral lipid envelope. Therefore, nursing by HCV-positive mothers is unlikely to play a major role in vertical transmission.
    The Journal of Infectious Diseases 09/2013; · 5.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28 days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2 days was observed at 37 °C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures.
    Journal of Viral Hepatitis 07/2013; 20(7):478-85. · 3.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.
    PLoS Pathogens 05/2013; 9(5):e1003355. · 8.14 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus genome replication is thought to occur in a membranous cellular compartment derived from the endoplasmic reticulum (ER). The molecular mechanisms by which these membrane-associated replication complexes are formed during HCV infection are only starting to be unraveled and both viral and cellular factors contribute to their formation. In this study, we describe the discovery of non-opioid sigma-1 receptor (S1R) as a cellular factor that mediates early steps of viral RNA replication. S1R is a cholesterol-binding protein that resides in lipid-rich areas of the ER and in mitochondria-associated ER membranes (MAMs). Several functions have been ascribed to this ER-resident chaperone, many of which are related to Ca(2+) signaling at the MAMs and lipid storage and trafficking. Downregulation of S1R expression by RNAi in Huh-7 cells leads to a proportional decrease in susceptibility to HCV infection, as shown by reduced HCV RNA accumulation and intra- and extracellular infectivity in single cycle infection experiments. Similar RNAi studies in persistently infected cells indicate that S1R-expression is not rate-limiting for persistent HCV RNA replication, as marked reduction in S1R in these cells does not lead to any decrease in HCV RNA or viral protein expression. However, subgenomic replicon transfection experiments indicate that S1R expression is rate-limiting for HCV RNA replication, without impairing primary translation. Overall, our data indicate that initial steps of HCV infection are regulated by S1R, a key component of MAMs, suggesting that these structures could serve as platforms for initial RNA replication during HCV infection.
    Journal of Virology 03/2013; · 5.08 Impact Factor
  • Zeitschrift für Gastroenterologie 01/2013; 51(01). · 1.41 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The hepatitis C virus (HCV) viroporin p7 is crucial for production of infectious viral progeny. However, its role in the viral replication cycle remains incompletely understood in part due to poor availability of p7-specific antibodies. To circumvent this obstacle, we inserted two consecutive HA-epitope tags at its N-terminus. HA-tagged p7 reduced peak virus titers ca. 10-fold and decreased kinetics of virus production compared to the wild type virus. However, HA- p7 rescued virus production of a mutant virus lacking p7, thus providing formal proof that the tag does not disrupt p7 function. In HCV-producing cells, p7 displayed a reticular staining pattern which co-localized with the HCV envelope glycoprotein 2 (E2), but also partially with viral non-structural proteins 2, 3 and 5A. Using co-immunoprecipitation, we confirmed a specific interaction between p7 and NS2, whereas we did not detect a stable interaction with core, E2 and NS5A. Moreover, we did not observe p7 incorporation into affinity-purified virus particles. Consistently, there was no evidence supporting a role of p7 in viral entry as an anti-HA antibody was not able to neutralize Jc1 virus produced from an HA-p7-tagged genome. Collectively, these findings highlight a stable interaction between p7 and NS2 which is likely crucial for production of infectious HCV particles. Use of this functional epitope-tagged p7 variant should facilitate the analysis of the final steps of the HCV replication cycle.
    Journal of Virology 11/2012; · 5.08 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: Contamination of blood products with hepatitis C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed. STUDY DESIGN AND METHODS: Inactivation studies were performed with cell culture-derived HCV and bovine viral diarrhea virus (BVDV), a model for HCV. Plasma units or PCs were spiked with high titers of cell culture-grown viruses. After treatment of the blood units with MB plus light (Theraflex MB-Plasma system, MacoPharma) or UVC (Theraflex UV-Platelets system, MacoPharma), residual viral infectivity was assessed using sensitive cell culture systems. RESULTS: HCV was sensitive to inactivation by both pathogen reduction procedures. HCV in plasma was efficiently inactivated by MB plus light below the detection limit already by 1/12 of the full light dose. HCV in PCs was inactivated by UVC irradiation with a reduction factor of more than 5 log. BVDV was less sensitive to the two pathogen reduction methods. CONCLUSIONS: Functional assays with human HCV offer an efficient tool to directly assess the inactivation capacity of pathogen reduction procedures. Pathogen reduction technologies such as MB plus light treatment and UVC irradiation have the potential to significantly reduce transfusion-transmitted HCV infections.
    Transfusion 08/2012; · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) cross-contamination from inanimate surfaces or objects has been implicated in transmission of HCV in health-care settings and among injection drug users. We established HCV-based carrier and drug transmission assays that simulate practical conditions to study inactivation and survival of HCV on inanimate surfaces. Studies were performed with authentic cell culture derived viruses. HCV was dried on steel discs and biocides were tested for their virucidal efficacy against HCV. Infectivity was determined by a limiting dilution assay. HCV stability was analyzed in a carrier assay for several days or in a drug transmission assay using a spoon as cooker. HCV can be dried and recovered efficiently in the carrier assay. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced infectivity of HCV to undetectable levels. Viral infectivity on inanimate surfaces was detectable in the presence of serum for up to 5 days, and temperatures of about 65-70°C were required to eliminate infectivity in the drug transmission assay. These findings are important for assessment of HCV transmission risks and should facilitate the definition of stringent public health interventions to prevent HCV infections.
    The Journal of Infectious Diseases 12/2011; 204(12):1830-8. · 5.85 Impact Factor
  • Source
    Clinical Infectious Diseases 08/2011; 53(9):963-4. · 9.37 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture-derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation.
    Hepatology 08/2011; 54(6):1947-55. · 12.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Patients with chronic hepatitis C virus (HCV) infection show an increased incidence of nervous system disorders such as chronic fatigue syndrome, depression and cognitive dysfunction. It is unclear whether this is because of HCV replication in the brain and in peripheral neuronal cells or to more indirect effects of HCV infection on the central or peripheral nervous system. The aim of this study was to investigate whether cells originating from these tissues are permissive for HCV cell entry, RNA replication and virus assembly. Among eight cell lines analysed, the human peripheral neuroblastoma cell line SKNMC expressed all HCV entry factors and was efficiently infected with HCV pseudoparticles (HCVpp) independent of the HCV genotype. All remaining cell types including human neuroblastoma and glioblastoma cell lines and microglial cells lacked expression of at least one host factor essential for HCV entry. When transfected with HCV luciferase reporter virus RNA, inoculated with HCV reporter viruses or challenged with high-titre cell culture-derived HCV, none of these cells supported detectable HCV RNA replication. Thus, in conclusion, this comprehensive screening did not reveal evidence directly strengthening the notion that HCV enters and replicates in the central nervous system. However, productive viral entry into the peripheral neuroblastoma cell line SKNMC indicates that HCV may penetrate into certain nonhepatic cell types which may serve as viral reservoirs and could modulate viral pathogenesis.
    Journal of Viral Hepatitis 08/2011; 18(8):562-70. · 3.08 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.
    PLoS Pathogens 04/2011; 7(4):e1002029. · 8.14 Impact Factor
  • Journal of Hepatology - J HEPATOL. 01/2011; 54.
  • Journal of Hepatology - J HEPATOL. 01/2011; 54.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In the absence of a cell culture system for propagation of the hepatitis C virus (HCV), the antiviral activity of disinfectants against HCV was extrapolated from studies with the bovine viral diarrhea virus. The recent development of an HCV infection system allowed the direct assessment of environmental stability and susceptibility to chemical disinfectants. Studies were performed using cell-culture grown HCV. Infectivity was determined by limiting dilutions. HCV RNA levels were analyzed by quantitative real-time polymerase chain reaction. Genome stability was determined by transfection of recovered RNA into Huh7.5 cells and immunostaining. HCV infectivity in a liquid environment was detectable for up to 5 month at lower temperatures. The risk of HCV infections may not accurately be reflected by determination of HCV RNA levels, because viral infectivity and HCV RNA copy numbers did not directly correlate. Different alcohols and commercially available antiseptics reduced the infectivity of HCV to undetectable levels. However, diluting the hand disinfectants abrogated the virucidal activity. This study assessed the environmental stability and susceptibility to chemical biocides of HCV. The results should be useful in defining rigorous disinfection protocols to prevent nosocomial transmission of HCV.
    The Journal of Infectious Diseases 06/2010; 201(12):1859-66. · 5.85 Impact Factor
  • Journal of Hepatology 04/2010; 52. · 9.86 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 (Delta HVR1) are less infectious, and separation by density gradients revealed that the population of Delta HVR1 virions comprises fewer particles with low density. Strikingly, Delta HVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, Delta HVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, Delta HVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection.
    Journal of Virology 03/2010; 84(11):5751-63. · 5.08 Impact Factor

Publication Stats

257 Citations
121.11 Total Impact Points

Institutions

  • 2010–2014
    • Hannover Medical School
      • Department of Gastroenterology, Hepatology and Endocrinology
      Hanover, Lower Saxony, Germany
  • 2013
    • National Center for Biotechnology (CNB)
      Madrid, Madrid, Spain
  • 2009–2013
    • TWINCORE
      • Institute for Experimental Virology
      Hanover, Lower Saxony, Germany
    • Universität Heidelberg
      • Department of Molecular Virology
      Heidelberg, Baden-Wuerttemberg, Germany
  • 2012
    • Helmholtz Centre for Infection Research
      Brunswyck, Lower Saxony, Germany