- Email: [email protected]
643 EXPRESSION OF HEPATITIS C VIRUS (HCV) RECEPTORS IN GRAFTS FROM HCV-INFECTED LIVER TRANSPLANT RECIPIENTS
POSTERS (ii) inhibition of viral polymerase activity by the 5’-triphosphate metabolite of ribavirin, (iii) induction of error catastrophe as a result of accumulation of mutations in the viral genome. Even if no direct relationship between ribavirin antiviral action and IMPDH inhibition has been demonstrated, the depletion of cellular GTP should result in an increased frequency of ribavirin triphosphate incorporation by viral polymerase due to lower intracellular concentration of its natural competitor. Aims: Relation between therapeutic potential of a nucleoside analogue and its anti-metabolite action remains difficult to demonstrate mainly because of the lack of investigation tools. The purpose of this study was to develop a range of assays to reveal nucleotide biosynthesis inhibition. Results: For a rapid evaluation of new nucleoside analogues as IMPDH inhibitors, we have developed an in vitro enzymatic assay where the synthesis of monophosphorylated form of nucleoside analogue (NA-MP) is provided by cloned human nucleoside kinases, and NA-MP is immediately tested for inhibition of human recombinant IMPDH. This assay has been validated with nucleoside analogues ribavirin and mizoribine. We have also developed original cell-based analytical approach in which 27 cellular ribo- and deoxyribonucleotides are extracted from cultured cells, separated by ion-pairing chromatography and quantified. This cellular assay, validated with several NA (ribavirin, aracytidine, gemcitabine) and known anti-metabolites (mycophenolic acid, leflunomide, hydroxyurea), provides a powerful tool for studying the effect of new nucleoside analogues on whole spectra of cellular purine and pyrimidine deoxy- and ribonucleotides. In addition, in regards with new antiviral molecules identified in HCV cell culture systems, our cellular assay allows to distinguish the molecules that directly acts on the viral proteins from others that inhibits the cell nucleotide biosynthesis. 642 CHARACTERISATION OF HEPATITIS C VIRUS ENTRY AND RNA REPLICATION IN CELLS OF CNS ORIGIN 1,2 B. Burgel ¨ , M. Friesland1,2 , A. Koch3 , M.P. Manns4 , H. Wedemeyer4 , K. Weissenborn5 , T. Pietschmann1,2 , E. Steinmann1,2 , S. Ciesek1,2,4 . 1 Department for Experimental Virology, Twincore, and Hannover Medical School, Hannover, 2 Helmholtz Centre for Infection Research, Braunschweig, 3 Department of Neuropathology, Charite – Universit¨ atsmedizin Berlin, Berlin, 4 Department of Gastroenterology, Hepatology and Endocrinology, 5 Department of Neurology, Hannover Medical School, Hannover, Germany E-mail: [email protected] Background and Aims: Patients with chronic hepatitis C virus (HCV) infection were reported to show nervous system disorders like chronic fatigue, depression and cognitive dysfunction. Until now, it is still unclear whether HCV infects and replicates in the brain or whether these symptoms are due to other processes associated with HCV infections. Previous studies could demonstrate the detection of HCV RNA and viral proteins in cells of autopsy brain tissue. The aim of this study is to investigate if cells of CNS origin are permissive for HCV cell entry, replication and virus assembly. Methods: Expression of the four essential HCV entry factors (CD81, SR-BI, Claudin-1, Occludin) was assessed in CNS cells by western blots and quantitative RT-PCR. HCV entry into the neuronal cells was tested with HCV pseudoparticles (HCVpp). Replication was analyzed by transfection of HCV reporter virus genomes into CNSderived cells. In addition, CNS cells were challenged with infectious cell culture derived HCV in the presence or absence of polymerase inhibitors. Results: All cells of CNS origin tested expressed detectable albeit variable quantities of HCV entry factor mRNAs. Moreover, CD81 protein was detected in all cell lines analyzed whereas all four factors together were only present in a single human neuroblastoma
cell line. These cells were permissive to HCVpp entry of different HCV genotypes. To analyze HCV RNA replication efficiency in this cell line, target cells were infected with high titer of Jc1 wildtype virus in the absence and presence of a NS5B inhibitor but no increase in HCV copy numbers over the inhibitor control could be detected. HCV RNA replication was below the detection limit in the different cell lines when HCV RNA was transfected into the cells with Luc-Jc1 reporter viruses. Conclusions: Several cells of CNS origin were analyzed for HCV entry and RNA replication to recapitulate HCV infections of the brain in vitro. A single cell line expressed all 4 HCV entry factors and was permissive for HCV entry, but not for RNA replication. However, low HCV RNA replication likely limits propagation in these cells and consequently viral spread and persistence in the central nervous system. 643 EXPRESSION OF HEPATITIS C VIRUS (HCV) RECEPTORS IN GRAFTS FROM HCV-INFECTED LIVER TRANSPLANT RECIPIENTS G. Crespo1 , L. Mensa1 , M. Gastinger2 , R. Miquel3 , S. Perez del Pulgar1 , S. Emerson4 , R.H. Purcell4 , X. Forns1 . 1 Liver Unit, Hospital Clinic de Barcelona, Barcelona, Spain; 2 Confocal and Biological Imaging Facility, National Institutes of Health, Bethesda, MD, USA; 3 Pathology Department, Hospital Clinic de Barcelona, Barcelona, Spain; 4 Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, MD, USA E-mail: [email protected] Background and Aims: Liver transplantation (LT) is a unique model to study the mechanisms of HCV entry into hepatocytes. After attachment to scavenger receptor B1 (SRB1), HCV binds CD81 and the virus-receptor complex is directed to tight junctions, where HCV interacts with claudin-1/occludin and enters the cells. Recent in vitro studies suggest significant changes in the patterns of claudin-1/occludin expression after HCV infection. The aims of this study were: 1) to characterize the patterns of expression of SRB1, claudin-1 and occludin in grafts from LT recipients and 2) to explore their potential changes before and after HCV infection. Patients and Methods: We included 42 HCV-infected liver transplant recipients (23 with mild hepatitis C recurrence and 19 with severe recurrence). SRB1, claudin-1 and occludin were detected in paraffin-embedded liver biopsies obtained during reperfusion, 3 and 12 months after LT. HCV receptors were stained by immunofluorescence and images were acquired by confocal microscopy (Leica SP5). Quantification and colocalization studies were performed with Imaris (Bitplane). Results: SRB1 expression was particularly abundant in the sinusoidal membrane. Claudin-1 and occludin expression was restricted to the apical (canalicular) membrane. There was a significant correlation between the amount of SRB1 expressed in reperfusion biopsies and the slope of HCV-RNA decay during the first 24 hours following LT (r= 0.6, p = 0.004). SRB1 levels remained stable after LT, whereas claudin-1 expression increased significantly between 3 and 12 months after LT (p < 0.01), both in patients with mild (p = 0.048) and severe (p = 0.025) hepatitis C recurrence. The increase in claudin-1 expression did not correlate with the severity of biochemical cholestasis or with HCV-RNA concentrations. Despite the increase in claudin-1 levels one year after HCV infection, the expression pattern remained unchanged and restricted to the apical membrane, where claudin-1 and occludin colocalized strongly (70–94%). Conclusions: SRB1 levels at time of LT may modulate the early HCV kinetics, probably by clearing circulating virions. An increase in claudin-1 levels one year after HCV infection did not translate into morphological changes in tight junctions; expression of claudin1/occludin remained restricted within the apical membrane of hepatocytes.
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cell line. These cells were permissive to HCVpp entry of different HCV genotypes. To analyze HCV RNA replication efficiency in this cell line, target cells were infected with high titer of Jc1 wildtype virus in the absence and presence of a NS5B inhibitor but no increase in HCV copy numbers over the inhibitor control could be detected. HCV RNA replication was below the detection limit in the different cell lines when HCV RNA was transfected into the cells with Luc-Jc1 reporter viruses. Conclusions: Several cells of CNS origin were analyzed for HCV entry and RNA replication to recapitulate HCV infections of the brain in vitro. A single cell line expressed all 4 HCV entry factors and was permissive for HCV entry, but not for RNA replication. However, low HCV RNA replication likely limits propagation in these cells and consequently viral spread and persistence in the central nervous system. 643 EXPRESSION OF HEPATITIS C VIRUS (HCV) RECEPTORS IN GRAFTS FROM HCV-INFECTED LIVER TRANSPLANT RECIPIENTS G. Crespo1 , L. Mensa1 , M. Gastinger2 , R. Miquel3 , S. Perez del Pulgar1 , S. Emerson4 , R.H. Purcell4 , X. Forns1 . 1 Liver Unit, Hospital Clinic de Barcelona, Barcelona, Spain; 2 Confocal and Biological Imaging Facility, National Institutes of Health, Bethesda, MD, USA; 3 Pathology Department, Hospital Clinic de Barcelona, Barcelona, Spain; 4 Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, MD, USA E-mail: [email protected] Background and Aims: Liver transplantation (LT) is a unique model to study the mechanisms of HCV entry into hepatocytes. After attachment to scavenger receptor B1 (SRB1), HCV binds CD81 and the virus-receptor complex is directed to tight junctions, where HCV interacts with claudin-1/occludin and enters the cells. Recent in vitro studies suggest significant changes in the patterns of claudin-1/occludin expression after HCV infection. The aims of this study were: 1) to characterize the patterns of expression of SRB1, claudin-1 and occludin in grafts from LT recipients and 2) to explore their potential changes before and after HCV infection. Patients and Methods: We included 42 HCV-infected liver transplant recipients (23 with mild hepatitis C recurrence and 19 with severe recurrence). SRB1, claudin-1 and occludin were detected in paraffin-embedded liver biopsies obtained during reperfusion, 3 and 12 months after LT. HCV receptors were stained by immunofluorescence and images were acquired by confocal microscopy (Leica SP5). Quantification and colocalization studies were performed with Imaris (Bitplane). Results: SRB1 expression was particularly abundant in the sinusoidal membrane. Claudin-1 and occludin expression was restricted to the apical (canalicular) membrane. There was a significant correlation between the amount of SRB1 expressed in reperfusion biopsies and the slope of HCV-RNA decay during the first 24 hours following LT (r= 0.6, p = 0.004). SRB1 levels remained stable after LT, whereas claudin-1 expression increased significantly between 3 and 12 months after LT (p < 0.01), both in patients with mild (p = 0.048) and severe (p = 0.025) hepatitis C recurrence. The increase in claudin-1 expression did not correlate with the severity of biochemical cholestasis or with HCV-RNA concentrations. Despite the increase in claudin-1 levels one year after HCV infection, the expression pattern remained unchanged and restricted to the apical membrane, where claudin-1 and occludin colocalized strongly (70–94%). Conclusions: SRB1 levels at time of LT may modulate the early HCV kinetics, probably by clearing circulating virions. An increase in claudin-1 levels one year after HCV infection did not translate into morphological changes in tight junctions; expression of claudin1/occludin remained restricted within the apical membrane of hepatocytes.
Journal of Hepatology 2010 vol. 52 | S183–S317
S251