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26 Heterologous T cell immunity during severe HCV infection
10
Thursday, April 15, 2004
to the wt virus. TLM deficient virus is trapped in the endosome. Based on these data we suggest the following model. After binding to the surface receptor(s?), the virus/receptor complex is internalized into the endosome. A proteolytic cleavage exposes the TLMs to the surface of the viral particle and enables the TLM-mediated escape of the processed particle in the cytoplasm.
cells ex-vivo. Only 4 weeks after the initial detection of NS3 1073-81, other HCV-specific CD8+ T cells started to be detected in vivo, gradually becoming dominant over the declining NS3 1073-81 specific CD8+ T cell population. Conclusions: Cross-reactive T cell immunity can influence the severity of liver immunopathology in HCV infection and contribute to shape the immunodominance of the HCV-specific T cell response.
25 HBV INFECTION INHIBITION BY PRE-S1 PEPTIDES DEPENDS ON THE N-TERMINAL ACYL MOIETY AND KEY AMINO ACIDS RECOGNISED BY NEUTRALISING ANTIBODIES
S. Urban 1 , P. Gripon 2 . 1 Molekulare Virologie, Otto Meyerhof Zentrum, Universität Heidelberg, Heidelberg, Germany; 2 INSERM U522, Hôpital De Pontchaillou, Rennes, France We have previously reported that myristoylated peptides representing an N-terminal preS1-domain of the HBV L-protein block HBV infection of human hepatocytes and HepaRG-cells. Infection interference is due to inactivation of a yet unknown cell-associated factor and requires the preS1 sequence of amino acids 19-48 (subtype ayw). In the present study, we extended our analysis and investigated (1) the role of the N-terminal 18 preS1 amino acids in infection inhibition, (2) the effect of the replacement of the N-terminal myristic acid by other fatty acids (e.g. palmitic acid, cholesterol and various saturated and unsaturated acyl residues) and (3) the importance of key amino acids within preS1-epitopes that are recognised by the neutralising antibodies MA18/7 (aa 20-23) and 5a19 (aa.26-32). Our results show that although inactive on their own, amino acids 2-19 are essential for infection interference by preS2-48myr, indicating a possible spacer function. Exchange of the N-terminal myristic acid by other hydrophobic residues did not abrogate infection inhibition, indicating no absolute requirement for this fatty acid. Moreover, the introduction of short deletions and single amino acids exchanges within preS19-48 allowed us to functionally map key amino acids required for inhibition. Our data shed light on the molecular mechanism of HBV entry into hepatocytes, as well as providing the possibility of modulating a peptide’s specific activities (including an increase of the ID50 to picomolar levels). Furthermore, these studies provide the basis for the development of new hepadnaviral entry inhibitors for therapeutic approaches.
26 HETEROLOGOUS T CELL IMMUNITY DURING SEVERE HCV INFECTION
S. Urbani 1 , P. Fisicaro 1 , B. Amadei 1 , G. Missale 1 , A. Bertoletti 2 , C. Ferrari 1 . 1 Laboratorio Immunopatologia Virale, Divisione Malattie Infettive Ed Epatologia, Azienda Ospedaliera Universitaria Di Parma, Parma, Italy; 2 Institute of Hepatology, Royal Free and University College Medical School, University College London, London, UK Background: Memory T cells specific for a previously encountered pathogen can be activated by a subsequent infection with an unrelated heterologous virus and can influence the clinical course of infection. Aim: We investigated the immunopathological mechanisms potentially responsible for 2 cases of severe acute hepatitis C characterized by elevated ALT (P1: 6540 IU/L; P 2: 3270 IU/L) and low protrombin time. Methods: Global analysis of HCV-specific T cell responses was performed longitudinally by Elispot analysis using a set of 600 overlapping 15 mer peptides covering the whole HCV sequence. HLA-A2-tetramers were used for detailed analysis of function and phenotype of selected CD8+ specificities. Results: Early after infection, a selective massive expansion of NS3 107381 specific CD8+ T cells, a CD8 population known to cross-react with an influenza virus immunodominant peptide, was observed in both patients. Frequency of NS3 1073-81 specific CD8+ T cells reached levels of 30% (P1) and 12% (P2) of total CD8 in the absence of other HCV-specific T cell responses. Virtually all NS3 1073-81 tetramer+ CD8 cells were HLA-DR+, CD45RA-, CCR7-, CD27+, perforin+, produced IFN-g and lysed target
27 IDENTIFICATION OF C-RAF1 AS A NOVEL BINDING PARTNER OF HCV REGULATORY PROTEIN NS5A
T. Buerckstuemmer, M. Kriegs, E. Hildt. Robert-Koch-Institut, Berlin, Germany Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein of 447 amino acids that has been shown to interact with a variety of cellular proteins that regulate signal transduction. However, few of these interactions provide an impact on understanding NS5A function in human hepatoma cell lines or in vivo. Using an affinity chromatography approach, c-Raf1 derived from human hepatoma cell lines was identified as a novel interaction partner of NS5A. This interaction was confirmed by coimmunoprecipitation experiments of hepatoma cell lysates. Moreover, we found that NS5A is colocalized with c-Raf1 in HuH-7 cells, as determined by confocal laser scanning microscopy. To characterize the interaction of NS5A with c-Raf1 in more detail, we generated deletion mutants of NS5A. c-Raf1 normally localized in the cytosol is translocated to the nucleus when coexpressed with N-terminal deletion mutants of NS5A that translocate to the nucleus. Since our data suggested that the serine/threonine kinase c-Raf1 interacts with NS5A, we investigated whether c-Raf1 is able to phosphorylate NS5A. In vitro phosphorylation experiments documented that c-Raf1 phosphorylates NS5A. A more detailed analysis revealed that the c-Raf1 dependent phosphorylation occurs in between amino acids 105 and 326 of NS5A. Finally, we showed that NS5A activates c-Raf1 as detected by increased ERK phosphorylation and immunocomplex assay after transient transfection of NS5A in human hepatoma cells (HepG2). To conclude, these experiments identify c-Raf1 as a novel binding partner of NS5A and provide new insights into NS5A dependent activation of signaling cascades.
28 INHIBITION OF HEPATITIS C VIRUS SUBGENOMIC REPLICATION BY CONSTITUTIVE TRANSPORT ELEMENT (CTE)-LINKED RIBOZYMES DIRECTED AGAINST THE HCV INTERNAL RIBOSOMAL ENTRY SITE (IRES) AND THE 3 UNTRANSLATED REGION (UTR)
D. Jarczak, M. Korf, M.P. Manns, M. Krueger. Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany Chronic HCV infection still is a clinically important disease with limited therapeutic options in a significant proportion of patients. Hairpin ribozymes (Rz) are small RNA molecules with endonuclease activity that can be engineered to specifically cleave RNA sequences. However, activity depends on Rz expression, target colocalization and cleavage site accessibility. CTE, an RNA motif has previously shown to interact with intracellular RNA helicases and might enhance cleavage activity by enhancement of RNA binding and unwinding activities of RNA helicase. CTE has also been shown to facilitate nuclear export, thereby potentially increasing cytoplasmatic Rz abundance and colocalization with HCV RNA substrate. The aim of our study was to investigate cleavage activities of endogenously expressed Rz linked to CTE on 5 - and 3 -UTR HCV RNA. Ribozymes were selected against highly conserved target sites within the 5 - and 3 -UTR HCV sequences. Retroviral vectors expressing Rz or RzCTE from a tRNA- or U6- promoter were transfected into Huh7 cells stably expressing monocistronic subgenomic HCV replicon. Western and northern blot analyses from transfected cultures demonstrated high ribozyme
Thursday, April 15, 2004
to the wt virus. TLM deficient virus is trapped in the endosome. Based on these data we suggest the following model. After binding to the surface receptor(s?), the virus/receptor complex is internalized into the endosome. A proteolytic cleavage exposes the TLMs to the surface of the viral particle and enables the TLM-mediated escape of the processed particle in the cytoplasm.
cells ex-vivo. Only 4 weeks after the initial detection of NS3 1073-81, other HCV-specific CD8+ T cells started to be detected in vivo, gradually becoming dominant over the declining NS3 1073-81 specific CD8+ T cell population. Conclusions: Cross-reactive T cell immunity can influence the severity of liver immunopathology in HCV infection and contribute to shape the immunodominance of the HCV-specific T cell response.
25 HBV INFECTION INHIBITION BY PRE-S1 PEPTIDES DEPENDS ON THE N-TERMINAL ACYL MOIETY AND KEY AMINO ACIDS RECOGNISED BY NEUTRALISING ANTIBODIES
S. Urban 1 , P. Gripon 2 . 1 Molekulare Virologie, Otto Meyerhof Zentrum, Universität Heidelberg, Heidelberg, Germany; 2 INSERM U522, Hôpital De Pontchaillou, Rennes, France We have previously reported that myristoylated peptides representing an N-terminal preS1-domain of the HBV L-protein block HBV infection of human hepatocytes and HepaRG-cells. Infection interference is due to inactivation of a yet unknown cell-associated factor and requires the preS1 sequence of amino acids 19-48 (subtype ayw). In the present study, we extended our analysis and investigated (1) the role of the N-terminal 18 preS1 amino acids in infection inhibition, (2) the effect of the replacement of the N-terminal myristic acid by other fatty acids (e.g. palmitic acid, cholesterol and various saturated and unsaturated acyl residues) and (3) the importance of key amino acids within preS1-epitopes that are recognised by the neutralising antibodies MA18/7 (aa 20-23) and 5a19 (aa.26-32). Our results show that although inactive on their own, amino acids 2-19 are essential for infection interference by preS2-48myr, indicating a possible spacer function. Exchange of the N-terminal myristic acid by other hydrophobic residues did not abrogate infection inhibition, indicating no absolute requirement for this fatty acid. Moreover, the introduction of short deletions and single amino acids exchanges within preS19-48 allowed us to functionally map key amino acids required for inhibition. Our data shed light on the molecular mechanism of HBV entry into hepatocytes, as well as providing the possibility of modulating a peptide’s specific activities (including an increase of the ID50 to picomolar levels). Furthermore, these studies provide the basis for the development of new hepadnaviral entry inhibitors for therapeutic approaches.
26 HETEROLOGOUS T CELL IMMUNITY DURING SEVERE HCV INFECTION
S. Urbani 1 , P. Fisicaro 1 , B. Amadei 1 , G. Missale 1 , A. Bertoletti 2 , C. Ferrari 1 . 1 Laboratorio Immunopatologia Virale, Divisione Malattie Infettive Ed Epatologia, Azienda Ospedaliera Universitaria Di Parma, Parma, Italy; 2 Institute of Hepatology, Royal Free and University College Medical School, University College London, London, UK Background: Memory T cells specific for a previously encountered pathogen can be activated by a subsequent infection with an unrelated heterologous virus and can influence the clinical course of infection. Aim: We investigated the immunopathological mechanisms potentially responsible for 2 cases of severe acute hepatitis C characterized by elevated ALT (P1: 6540 IU/L; P 2: 3270 IU/L) and low protrombin time. Methods: Global analysis of HCV-specific T cell responses was performed longitudinally by Elispot analysis using a set of 600 overlapping 15 mer peptides covering the whole HCV sequence. HLA-A2-tetramers were used for detailed analysis of function and phenotype of selected CD8+ specificities. Results: Early after infection, a selective massive expansion of NS3 107381 specific CD8+ T cells, a CD8 population known to cross-react with an influenza virus immunodominant peptide, was observed in both patients. Frequency of NS3 1073-81 specific CD8+ T cells reached levels of 30% (P1) and 12% (P2) of total CD8 in the absence of other HCV-specific T cell responses. Virtually all NS3 1073-81 tetramer+ CD8 cells were HLA-DR+, CD45RA-, CCR7-, CD27+, perforin+, produced IFN-g and lysed target
27 IDENTIFICATION OF C-RAF1 AS A NOVEL BINDING PARTNER OF HCV REGULATORY PROTEIN NS5A
T. Buerckstuemmer, M. Kriegs, E. Hildt. Robert-Koch-Institut, Berlin, Germany Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein of 447 amino acids that has been shown to interact with a variety of cellular proteins that regulate signal transduction. However, few of these interactions provide an impact on understanding NS5A function in human hepatoma cell lines or in vivo. Using an affinity chromatography approach, c-Raf1 derived from human hepatoma cell lines was identified as a novel interaction partner of NS5A. This interaction was confirmed by coimmunoprecipitation experiments of hepatoma cell lysates. Moreover, we found that NS5A is colocalized with c-Raf1 in HuH-7 cells, as determined by confocal laser scanning microscopy. To characterize the interaction of NS5A with c-Raf1 in more detail, we generated deletion mutants of NS5A. c-Raf1 normally localized in the cytosol is translocated to the nucleus when coexpressed with N-terminal deletion mutants of NS5A that translocate to the nucleus. Since our data suggested that the serine/threonine kinase c-Raf1 interacts with NS5A, we investigated whether c-Raf1 is able to phosphorylate NS5A. In vitro phosphorylation experiments documented that c-Raf1 phosphorylates NS5A. A more detailed analysis revealed that the c-Raf1 dependent phosphorylation occurs in between amino acids 105 and 326 of NS5A. Finally, we showed that NS5A activates c-Raf1 as detected by increased ERK phosphorylation and immunocomplex assay after transient transfection of NS5A in human hepatoma cells (HepG2). To conclude, these experiments identify c-Raf1 as a novel binding partner of NS5A and provide new insights into NS5A dependent activation of signaling cascades.
28 INHIBITION OF HEPATITIS C VIRUS SUBGENOMIC REPLICATION BY CONSTITUTIVE TRANSPORT ELEMENT (CTE)-LINKED RIBOZYMES DIRECTED AGAINST THE HCV INTERNAL RIBOSOMAL ENTRY SITE (IRES) AND THE 3 UNTRANSLATED REGION (UTR)
D. Jarczak, M. Korf, M.P. Manns, M. Krueger. Department of Gastroenterology, Hepatology and Endocrinology, Medizinische Hochschule Hannover, Hannover, Germany Chronic HCV infection still is a clinically important disease with limited therapeutic options in a significant proportion of patients. Hairpin ribozymes (Rz) are small RNA molecules with endonuclease activity that can be engineered to specifically cleave RNA sequences. However, activity depends on Rz expression, target colocalization and cleavage site accessibility. CTE, an RNA motif has previously shown to interact with intracellular RNA helicases and might enhance cleavage activity by enhancement of RNA binding and unwinding activities of RNA helicase. CTE has also been shown to facilitate nuclear export, thereby potentially increasing cytoplasmatic Rz abundance and colocalization with HCV RNA substrate. The aim of our study was to investigate cleavage activities of endogenously expressed Rz linked to CTE on 5 - and 3 -UTR HCV RNA. Ribozymes were selected against highly conserved target sites within the 5 - and 3 -UTR HCV sequences. Retroviral vectors expressing Rz or RzCTE from a tRNA- or U6- promoter were transfected into Huh7 cells stably expressing monocistronic subgenomic HCV replicon. Western and northern blot analyses from transfected cultures demonstrated high ribozyme