- Email: [email protected]
317 DYSREGULATION OF GLUCONEOGENESIS IN CHRONIC HEPATITIS C: THE ROLE OF PROTEIN PHOSPHATASE 2A
05b: VIRAL HEPATITIS − b) HEPATITIS C − EXPERIMENTAL (VIROLOGY) interval. After the second DC-injection, tumors were analyzed by flow cytometry. Results: Co-culture with Ad-CD40L-transduced DC enhanced significantly the cytotoxicity against Hepa129-cells of splenocytes in comparison to co-culture with Ad-IL-2 or Ad-LacZ-transduced DC (p < 0.05). Combination of CD40L/IL-2-expressing DC inhibited the tumor-growth significantly compared to mice treated with control DC or IL-2-DC (p < 0.05). However, the antitumoral activity of the combination was not stronger compared to DC transduced only with Ad-CD40L. Furthermore, CD40L-DC showed even a stronger inhibition of tumor growth than the combination of CD40L/IL-2-DC: the mean tumor volume was 361.4±118.5 mm3 in CD40L-DC-treated animals vs. 868.2±510.5 in CD40L/IL-2- or 1805±309.7 mm3 in LacZ-DC-treated mice. Flow cytometry of tumors treated with CD40L-expressing DC revealed higher tumor-infiltration with CD4+-, CD8+-T-cells and NK-cells than tumors treated with CD40L/IL-2 DC. Conclusions: The combination of CD40L/IL-2-expressing DC can significantly inhibit the tumor-growth, activating both acquired and innate immunity. However, this effect seemed not to be synergistic, as treatments with CD40L-DC seemed to have even stronger antitumoral effects than the combination of CD40L- and IL-2-DC. These data underline the potential of the co-stimulation on CD40/CD40L-interaction in DC-based approaches against tumor disease.
05b: VIRAL HEPATITIS − b) HEPATITIS C − EXPERIMENTAL (VIROLOGY)
315 HCV-RNA DECLINE IN THE FIRST 48 HOURS IDENTIFIES HCV MONOINFECTED AND HCV/HIV COINFECTED PATIENTS WITH RVR AT WEEK 4 OF PEGINTERFERON-ALPHA-2A/RIBAVIRIN THERAPY J.E. Arends1 , J. Cohen Stuart2 , L.C. Baak3 , M.E. van der Ende4 , K.J. van Erpecum5 , G.J. Boland2 , D. van Baarle1,6 , I.M. Hoepelman1 . 1 Internal Medicine and Infectious Diseases, 2 Medical Microbiology, University Medical Center Utrecht, Utrecht, 3 Gastroenterology, Onze Lieve Vrouwe Gasthuis, Amsterdam, 4 Internal Medicine and Infectious Diseases, Erasmus Medical Center, Rotterdam, 5 Gastroenterology, 6 Immunology, University Medical Center Utrecht, Utrecht, The Netherlands E-mail: [email protected] Background: During peginterferon-alpha-2a/ribavirin therapy, plasma hepatitis C virus (HCV)-RNA decreases with a rapid first phase and a slower second phase. We compared the first phase viral load decrease and viral slope in patients with a rapid viral response (RVR, i.e. PCR < 50 IU/ml at week 4) with patients not achieving a RVR. Methods: From 23 HCV infected (14 mono-infected and 9 HCV/HIV coinfected) genotype 1 or 4 positive peginterferon alfa-2a/ribavirin treated patients, plasma HCV-RNA was determined at baseline, 48 hours, weeks 1, 2, 4, 8 and 12. The first phase HCV viral load decrease (D0−48 ), the first phase slope (l1 ) and the efficiency factor (e) were determined.
Viral decrease.
S123
Results: Eight patients with a RVR showed both a greater D0−48 and l1 (−1.49log 10 ±0.57 IU/mL and −1.72/day±0.66) compared to 15 nonRVR patients (−0.71log 10 ±0.62 IU/mL and −0.85/day±0.70; p = 0.037). A greater D0−48 and l1 was also seen in patients achieving a sustained viral response (SVR) compared to patients without a SVR (p = 0.015). e was significantly higher in RVR-patients (98%±6.7) compared to nonRVR-patients (81%±28.5; p = 0.037). Conclusion: In the first 48 hours after start of therapy, patients with a RVR have a larger viral load decrease, steeper viral slope and a higher efficiency factor when compared to non-RVR patients. 316 IN VIVO ENDOPLASMIC RETICULUM STRESS IN PATIENTS WITH CHRONIC HEPATITIS C T. Asselah1 , I. Bi`eche2 , A. Mansouri3 , I. Laurendeau2 , D. CazalsHatem4 , G. Feldmann5 , P. Bedossa4 , V. Paradis4 , M. Martinot-Peignoux6 , D. Lebrec1 , C. Guichard5 , E. Ogier-Denis3 , M. Vidaud7 , Z. Tellier8 , P. Marcellin1 , R. Moreau6 . 1 Service d’H´epatologie, Hˆopital Beaujon, Clichy, 2 INSERM U745, 3 INSERM U773F, Paris, 4 Service d’ AnatomiePathologie, Clichy, 5 INSERM U773, Paris, 6 INSERM U773, 7 Service de Biochimie, Clichy, 8 Laboratoire Fran¸cais du Fractionnement, Courtaboeuf, France E-mail: [email protected] Background and Aims: In hepatocytes, the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER-resident stress sensors ATF-6, IRE1 and PERK. The UPR protect cells by inducing genes that enhance degradation of misfolded proteins, and reducing the synthesis of new proteins (via PERK-induced phosphorylation of eIF2alpha). Thus, we investigated in vivo ER stress and the UPR in liver samples from untreated patients with mild chronic hepatitis C (CHC). Methods: Liver samples (normal liver and mild HCV-related fibrosis) were studied by electron microscopy, western blotting and real-time quantitative RT-PCR. Results: In mild CHC, electron microscopy identified features suggesting ER stress in hepatocytes. Moreover, the ER stress sensors ATF-6, IRE1, and PERK were activated in mild fibrosis. However, there was no significant induction of UPR-responsive genes XBP1, ATF6, CREB1L1, ATF4, HSPA5/BiP/GRP78, HSP90B1/GRP94, CANX , CALR, ATP2A2, EDEM1, DDIT3/CHOP while PPP1R15A/GADD34 (involved in eIF2alpha dephosphorylation) was significantly downregulated. Conclusions: Livers from patients with mild CHC exhibit ER stress, activation of ATF-6, IRE1, and PERK without induction of UPR-responsive genes. Our findings suggest that HCV inhibits the induction of UPR, in particular to prevent degradation of misfolded viral proteins. 317 DYSREGULATION OF GLUCONEOGENESIS IN CHRONIC HEPATITIS C: THE ROLE OF PROTEIN PHOSPHATASE 2A C. Bernsmeier1,2 , F.H.T. Duong1 , S. Lin1 , M.H. Heim1,2 . 1 Hepatology Laboratory, Department of Biomedicine, 2 Department of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland E-mail: [email protected] Background and Aims: Insulin resistance and type 2 diabetes mellitus are metabolic disorders induced by the hepatitis C virus. They are risk factors for fibrosis progression and non-response to antiviral therapy in chronic hepatitis C (CHC). The molecular mechanisms responsible for insulin resistance in CHC remain to be elucidated. We have previously shown that HCV induced overexpression of Protein phosphatase 2 A (PP2A) in the liver inhibits phosphorylation of AMPK and PKB/Akt, two important kinases of the insulin signalling cascade. The aim was to study glucose homeostasis and gluconeogenetic gene regulation in HCV transgenic mice in order to understand the role of PP2A in glucose homeostasis dysregulation. Methods: We used HCV transgenic (B6HCV) mice to study glucose metabolism and the expression of gluconeogenetic key enzymes in the
S124
Poster Session − Thursday, April 23
liver. Insulin sensitivity was analysed by measuring fasting glucose and insulin levels (for calculation of HOMA) and by glucose- and insulin tolerance tests. We studied phosphorylation of Foxo1 at Ser256 by Western Blot and expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phophatase (G6Pase), two crucial gluconeogenetic key enzymes, and PGC1a, their major transcriptional coactivator, by quantitative RTPCR. Results: Compared to C57BL6 mice, B6HCV mice have elevated fasting glucose (6.5 mmol/l vs. 4.9 mmol/l), fasting insulin (32.4 mIU/ml vs. 14.7 mIU/ml) and HOMA index (8.4 vs. 2.8). Glucose and insulin tolerance tests were pathologic. S256Foxo1 was hypophosphorylated in B6HCV mice and PGC1a expression was significantly upregulated. Also upregulation of PEPCK and G6Pase was observed. Conclusion: The transgenic expression of a HCV genotype 1b genome in hepatocytes results in a dysregulated expression of gluconeogenetic key enzymes and leads to systemic insulin resistance by a mechanism involving PP2A upregulation and interference with insulin signalling. 318 THE HEPATITIS C VIRUS ENHANCES ACTIVATION OF THE EGF-RECEPTOR AND AKT-ACTIVITY BY NS3/4A-MEDIATED CLEAVAGE OF THE T-CELL PROTEIN TYROSINE PHOSPHATASE E. Brennd¨orfer1,2 , J. Karthe1 , L. Frelin2 , P. Cebula1 , A. Erhardt1 , H. Hengel3 , R. Bartenschlager4 , M. S¨allberg2 , D. H¨aussinger1 , J. Bode1 . 1 Department of Gastroenterology, Heaptology and Infectiology, Heinrich-Heine Universit¨at, D¨usseldorf, Germany; 2 Division of Clinical Microbiology, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden; 3 Institute of Virology, University Hospital D¨usseldorf, D¨usseldorf, 4 Department of Molecular Virology, University Hospital, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background and Aims: The hepatitis C virus (HCV) is worldwide a major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication on the one hand and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. Material and Method: Cell lines harboring the subgenomic replicon of HCV and cell lines stably expressing NS3/4A as well as liver tissue from transgenic animals with liver specific expression of NS3/4A and tissue samples from patients with chronic HCV infection were analyzed. Results: In this study we describe a novel mechanism by which HCV enhances activation of the EGFR/Akt pathway, which is known to mediate important cellular growth and differentiation signals, through a NS3/4A protease-mediated downregulation of the ubiquitously expressed tyrosine phosphatase TC-PTP. NS3/4A is demonstrated to cleave TC-PTP in vitro at two cleavage sites, resulting in the appearance of two fragments. The in vivo relevance of this finding is supported by the fact that downregulation of TC-PTP protein expression could also be demonstrated in HCV infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. TC-PTP is important for negative regulation of the EGFR and as indicated by data presented herein also indirectly controls the basal activity of Akt. This might explain why HCV not only sensitizes for ligand-induced EGFR activation but also causes a constitutive activation of Akt, which is demonstrated to be required for sufficient replication of HCV. Conclusion: Our findings support the notion that therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects further diminishing viral replication. The NS3/4A inhibition reduces the processing of the viral polyprotein but also deprives HCV of modulating EGFR signaling to its own purposes.
319 DEVELOPMENT OF A PLATFORM TO DETECT DRUG RESISTANCE MUTATIONS IN THE NON-STRUCTURAL PROTEIN REGION OF THE HEPATITIS C VIRUS GENOTYPES 1, 2, 3, AND 4 S. Dumont1 , B. Fevery1 , K. Van den Brande1 , V. Baumer1 , H. Ceulemans2 , H. De Wolf1 , L.J. Stuyver1 , D. Koletzki1 . 1 Virco BVBA, 2 Tibotec BVBA, Mechelen, Belgium E-mail: [email protected] Background and Aims: Given the HCV quasispecies diversity driven by high viral load and high error rate during RNA replication, it will be important to detect sequence variants from serum of HCV-infected patients. With the advent of direct antivirals targeting HCV, the detection of drug-resistance associated mutations may be of increasing importance. We have developed integrated assays for HCV genotypic analysis. Methods: Using clinical isolates representing all six genotypes, we developed two test systems; an NS5B-based subtyping assay, and a set of subtype-specific genotyping assays in the following target regions: (1) complete NS3/4A and the N-terminal 181aa of NS3 (resistance to protease inhibitors), (2) complete NS5B (resistance to polymerase inhibitors), and (3) complete NS4B/5A, for subtypes 1a, 1b, 2a, 2b, 3a, 4a and 4d. Two software tools were designed: (1) a BLAST-based subtyper and (2) a Hidden Markov Model-based (HMM) alignment tool. The genotypic assays were optimized and validated to support high-throughput processing in a routine operational setting. Results: Approximately 600 clinical samples representing all six genotypes were tested using the NS5B-based subtyping assay. The subtypespecific genotyping assays were evaluated by testing sets of clinical samples. The HMM alignment software was used to automatically align sample sequences to their respective subtype references.
Analytical sensitivity (AS) @IU/ml (n)
NS5Bsubt. NS3/4A 181aaNS3 NS4B/5A NS5B
CS (n)
genotype 1a/b
genotype 2a/b
genotype 3a
genotype 4a/d
overall
86%@3log 88%@4log 86%@3log 90%@2log 95%@4log
86%@3log 67%@4log 67%@3log NA 75%@2log
90%@3log 90%@2log 90%@2log 80%@2log 88%@4log
70%@4log 88%@3log 88%@4log 70%@2log 80%@2log
91% 94% 97% 95% 95%
(14) (7) (14) (20) (20)
(7) (3) (6) (4)*
(10) (10) (10) (10) (8)
(10) (8) (8) (10) (10)
(603) (273) (274) (249) (260)
CS, clinical sensitivity; *only 2b.
Conclusions: We have demonstrated that the NS5B sequence-based subtyping assay detects all six genotypes and discriminates between the different subtypes with high CS and AS. Genotyping assays covering the complete coding region from NS3 to NS5B have been developed on a large panel of clinical samples including protocols for subtypes 1a, 1b, 2a, 2b, 3a, 4a and 4d. The performance characteristics of the assays make them suitable for potential use in clinical settings.
320 COMPETITION BETWEEN DIVERGENT HEPATITIS C VIRUS STRAINS DRIVEN BY INTERNAL RIBOSOME ENTRY SITE EFFICIENCY IN LIVER AND B-CELLS T. Durand1 , H. Colman1 , G. Diliberto2 , A. Cammas3 , P. Marcellin4 , S. Vagner3 , C. F´eray1 . 1 INSERM 948 University Nantes, Nantes, 2 Inserm U773, Clichy, 3 INSERM 758, Lyon, 4 INSERM 773, Clichy, France E-mail: [email protected] Background: Hepatitis C virus replicates in hepatocytes but also in B-cells. In some subjects exposed to repeated infections by HCV, we previously showed that B-cells harbor HCV strains not detected in plasma or in the liver (Di Liberto, Gastroenterology, 2006). These strains have divergent structural and non structural proteins and also divergent internal ribosomal entry sites (IRES). This untranslated part of RNA regulates the translation of the viral polyprotein and its efficiency usually varies according to cell types. The IRES could determine the competition between HCV variants
05b: VIRAL HEPATITIS − b) HEPATITIS C − EXPERIMENTAL (VIROLOGY)
315 HCV-RNA DECLINE IN THE FIRST 48 HOURS IDENTIFIES HCV MONOINFECTED AND HCV/HIV COINFECTED PATIENTS WITH RVR AT WEEK 4 OF PEGINTERFERON-ALPHA-2A/RIBAVIRIN THERAPY J.E. Arends1 , J. Cohen Stuart2 , L.C. Baak3 , M.E. van der Ende4 , K.J. van Erpecum5 , G.J. Boland2 , D. van Baarle1,6 , I.M. Hoepelman1 . 1 Internal Medicine and Infectious Diseases, 2 Medical Microbiology, University Medical Center Utrecht, Utrecht, 3 Gastroenterology, Onze Lieve Vrouwe Gasthuis, Amsterdam, 4 Internal Medicine and Infectious Diseases, Erasmus Medical Center, Rotterdam, 5 Gastroenterology, 6 Immunology, University Medical Center Utrecht, Utrecht, The Netherlands E-mail: [email protected] Background: During peginterferon-alpha-2a/ribavirin therapy, plasma hepatitis C virus (HCV)-RNA decreases with a rapid first phase and a slower second phase. We compared the first phase viral load decrease and viral slope in patients with a rapid viral response (RVR, i.e. PCR < 50 IU/ml at week 4) with patients not achieving a RVR. Methods: From 23 HCV infected (14 mono-infected and 9 HCV/HIV coinfected) genotype 1 or 4 positive peginterferon alfa-2a/ribavirin treated patients, plasma HCV-RNA was determined at baseline, 48 hours, weeks 1, 2, 4, 8 and 12. The first phase HCV viral load decrease (D0−48 ), the first phase slope (l1 ) and the efficiency factor (e) were determined.
Viral decrease.
S123
Results: Eight patients with a RVR showed both a greater D0−48 and l1 (−1.49log 10 ±0.57 IU/mL and −1.72/day±0.66) compared to 15 nonRVR patients (−0.71log 10 ±0.62 IU/mL and −0.85/day±0.70; p = 0.037). A greater D0−48 and l1 was also seen in patients achieving a sustained viral response (SVR) compared to patients without a SVR (p = 0.015). e was significantly higher in RVR-patients (98%±6.7) compared to nonRVR-patients (81%±28.5; p = 0.037). Conclusion: In the first 48 hours after start of therapy, patients with a RVR have a larger viral load decrease, steeper viral slope and a higher efficiency factor when compared to non-RVR patients. 316 IN VIVO ENDOPLASMIC RETICULUM STRESS IN PATIENTS WITH CHRONIC HEPATITIS C T. Asselah1 , I. Bi`eche2 , A. Mansouri3 , I. Laurendeau2 , D. CazalsHatem4 , G. Feldmann5 , P. Bedossa4 , V. Paradis4 , M. Martinot-Peignoux6 , D. Lebrec1 , C. Guichard5 , E. Ogier-Denis3 , M. Vidaud7 , Z. Tellier8 , P. Marcellin1 , R. Moreau6 . 1 Service d’H´epatologie, Hˆopital Beaujon, Clichy, 2 INSERM U745, 3 INSERM U773F, Paris, 4 Service d’ AnatomiePathologie, Clichy, 5 INSERM U773, Paris, 6 INSERM U773, 7 Service de Biochimie, Clichy, 8 Laboratoire Fran¸cais du Fractionnement, Courtaboeuf, France E-mail: [email protected] Background and Aims: In hepatocytes, the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) causes ER stress and the unfolded protein response (UPR), mediated by the ER-resident stress sensors ATF-6, IRE1 and PERK. The UPR protect cells by inducing genes that enhance degradation of misfolded proteins, and reducing the synthesis of new proteins (via PERK-induced phosphorylation of eIF2alpha). Thus, we investigated in vivo ER stress and the UPR in liver samples from untreated patients with mild chronic hepatitis C (CHC). Methods: Liver samples (normal liver and mild HCV-related fibrosis) were studied by electron microscopy, western blotting and real-time quantitative RT-PCR. Results: In mild CHC, electron microscopy identified features suggesting ER stress in hepatocytes. Moreover, the ER stress sensors ATF-6, IRE1, and PERK were activated in mild fibrosis. However, there was no significant induction of UPR-responsive genes XBP1, ATF6, CREB1L1, ATF4, HSPA5/BiP/GRP78, HSP90B1/GRP94, CANX , CALR, ATP2A2, EDEM1, DDIT3/CHOP while PPP1R15A/GADD34 (involved in eIF2alpha dephosphorylation) was significantly downregulated. Conclusions: Livers from patients with mild CHC exhibit ER stress, activation of ATF-6, IRE1, and PERK without induction of UPR-responsive genes. Our findings suggest that HCV inhibits the induction of UPR, in particular to prevent degradation of misfolded viral proteins. 317 DYSREGULATION OF GLUCONEOGENESIS IN CHRONIC HEPATITIS C: THE ROLE OF PROTEIN PHOSPHATASE 2A C. Bernsmeier1,2 , F.H.T. Duong1 , S. Lin1 , M.H. Heim1,2 . 1 Hepatology Laboratory, Department of Biomedicine, 2 Department of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland E-mail: [email protected] Background and Aims: Insulin resistance and type 2 diabetes mellitus are metabolic disorders induced by the hepatitis C virus. They are risk factors for fibrosis progression and non-response to antiviral therapy in chronic hepatitis C (CHC). The molecular mechanisms responsible for insulin resistance in CHC remain to be elucidated. We have previously shown that HCV induced overexpression of Protein phosphatase 2 A (PP2A) in the liver inhibits phosphorylation of AMPK and PKB/Akt, two important kinases of the insulin signalling cascade. The aim was to study glucose homeostasis and gluconeogenetic gene regulation in HCV transgenic mice in order to understand the role of PP2A in glucose homeostasis dysregulation. Methods: We used HCV transgenic (B6HCV) mice to study glucose metabolism and the expression of gluconeogenetic key enzymes in the
S124
Poster Session − Thursday, April 23
liver. Insulin sensitivity was analysed by measuring fasting glucose and insulin levels (for calculation of HOMA) and by glucose- and insulin tolerance tests. We studied phosphorylation of Foxo1 at Ser256 by Western Blot and expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phophatase (G6Pase), two crucial gluconeogenetic key enzymes, and PGC1a, their major transcriptional coactivator, by quantitative RTPCR. Results: Compared to C57BL6 mice, B6HCV mice have elevated fasting glucose (6.5 mmol/l vs. 4.9 mmol/l), fasting insulin (32.4 mIU/ml vs. 14.7 mIU/ml) and HOMA index (8.4 vs. 2.8). Glucose and insulin tolerance tests were pathologic. S256Foxo1 was hypophosphorylated in B6HCV mice and PGC1a expression was significantly upregulated. Also upregulation of PEPCK and G6Pase was observed. Conclusion: The transgenic expression of a HCV genotype 1b genome in hepatocytes results in a dysregulated expression of gluconeogenetic key enzymes and leads to systemic insulin resistance by a mechanism involving PP2A upregulation and interference with insulin signalling. 318 THE HEPATITIS C VIRUS ENHANCES ACTIVATION OF THE EGF-RECEPTOR AND AKT-ACTIVITY BY NS3/4A-MEDIATED CLEAVAGE OF THE T-CELL PROTEIN TYROSINE PHOSPHATASE E. Brennd¨orfer1,2 , J. Karthe1 , L. Frelin2 , P. Cebula1 , A. Erhardt1 , H. Hengel3 , R. Bartenschlager4 , M. S¨allberg2 , D. H¨aussinger1 , J. Bode1 . 1 Department of Gastroenterology, Heaptology and Infectiology, Heinrich-Heine Universit¨at, D¨usseldorf, Germany; 2 Division of Clinical Microbiology, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden; 3 Institute of Virology, University Hospital D¨usseldorf, D¨usseldorf, 4 Department of Molecular Virology, University Hospital, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany E-mail: [email protected] Background and Aims: The hepatitis C virus (HCV) is worldwide a major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication on the one hand and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. Material and Method: Cell lines harboring the subgenomic replicon of HCV and cell lines stably expressing NS3/4A as well as liver tissue from transgenic animals with liver specific expression of NS3/4A and tissue samples from patients with chronic HCV infection were analyzed. Results: In this study we describe a novel mechanism by which HCV enhances activation of the EGFR/Akt pathway, which is known to mediate important cellular growth and differentiation signals, through a NS3/4A protease-mediated downregulation of the ubiquitously expressed tyrosine phosphatase TC-PTP. NS3/4A is demonstrated to cleave TC-PTP in vitro at two cleavage sites, resulting in the appearance of two fragments. The in vivo relevance of this finding is supported by the fact that downregulation of TC-PTP protein expression could also be demonstrated in HCV infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. TC-PTP is important for negative regulation of the EGFR and as indicated by data presented herein also indirectly controls the basal activity of Akt. This might explain why HCV not only sensitizes for ligand-induced EGFR activation but also causes a constitutive activation of Akt, which is demonstrated to be required for sufficient replication of HCV. Conclusion: Our findings support the notion that therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects further diminishing viral replication. The NS3/4A inhibition reduces the processing of the viral polyprotein but also deprives HCV of modulating EGFR signaling to its own purposes.
319 DEVELOPMENT OF A PLATFORM TO DETECT DRUG RESISTANCE MUTATIONS IN THE NON-STRUCTURAL PROTEIN REGION OF THE HEPATITIS C VIRUS GENOTYPES 1, 2, 3, AND 4 S. Dumont1 , B. Fevery1 , K. Van den Brande1 , V. Baumer1 , H. Ceulemans2 , H. De Wolf1 , L.J. Stuyver1 , D. Koletzki1 . 1 Virco BVBA, 2 Tibotec BVBA, Mechelen, Belgium E-mail: [email protected] Background and Aims: Given the HCV quasispecies diversity driven by high viral load and high error rate during RNA replication, it will be important to detect sequence variants from serum of HCV-infected patients. With the advent of direct antivirals targeting HCV, the detection of drug-resistance associated mutations may be of increasing importance. We have developed integrated assays for HCV genotypic analysis. Methods: Using clinical isolates representing all six genotypes, we developed two test systems; an NS5B-based subtyping assay, and a set of subtype-specific genotyping assays in the following target regions: (1) complete NS3/4A and the N-terminal 181aa of NS3 (resistance to protease inhibitors), (2) complete NS5B (resistance to polymerase inhibitors), and (3) complete NS4B/5A, for subtypes 1a, 1b, 2a, 2b, 3a, 4a and 4d. Two software tools were designed: (1) a BLAST-based subtyper and (2) a Hidden Markov Model-based (HMM) alignment tool. The genotypic assays were optimized and validated to support high-throughput processing in a routine operational setting. Results: Approximately 600 clinical samples representing all six genotypes were tested using the NS5B-based subtyping assay. The subtypespecific genotyping assays were evaluated by testing sets of clinical samples. The HMM alignment software was used to automatically align sample sequences to their respective subtype references.
Analytical sensitivity (AS) @IU/ml (n)
NS5Bsubt. NS3/4A 181aaNS3 NS4B/5A NS5B
CS (n)
genotype 1a/b
genotype 2a/b
genotype 3a
genotype 4a/d
overall
86%@3log 88%@4log 86%@3log 90%@2log 95%@4log
86%@3log 67%@4log 67%@3log NA 75%@2log
90%@3log 90%@2log 90%@2log 80%@2log 88%@4log
70%@4log 88%@3log 88%@4log 70%@2log 80%@2log
91% 94% 97% 95% 95%
(14) (7) (14) (20) (20)
(7) (3) (6) (4)*
(10) (10) (10) (10) (8)
(10) (8) (8) (10) (10)
(603) (273) (274) (249) (260)
CS, clinical sensitivity; *only 2b.
Conclusions: We have demonstrated that the NS5B sequence-based subtyping assay detects all six genotypes and discriminates between the different subtypes with high CS and AS. Genotyping assays covering the complete coding region from NS3 to NS5B have been developed on a large panel of clinical samples including protocols for subtypes 1a, 1b, 2a, 2b, 3a, 4a and 4d. The performance characteristics of the assays make them suitable for potential use in clinical settings.
320 COMPETITION BETWEEN DIVERGENT HEPATITIS C VIRUS STRAINS DRIVEN BY INTERNAL RIBOSOME ENTRY SITE EFFICIENCY IN LIVER AND B-CELLS T. Durand1 , H. Colman1 , G. Diliberto2 , A. Cammas3 , P. Marcellin4 , S. Vagner3 , C. F´eray1 . 1 INSERM 948 University Nantes, Nantes, 2 Inserm U773, Clichy, 3 INSERM 758, Lyon, 4 INSERM 773, Clichy, France E-mail: [email protected] Background: Hepatitis C virus replicates in hepatocytes but also in B-cells. In some subjects exposed to repeated infections by HCV, we previously showed that B-cells harbor HCV strains not detected in plasma or in the liver (Di Liberto, Gastroenterology, 2006). These strains have divergent structural and non structural proteins and also divergent internal ribosomal entry sites (IRES). This untranslated part of RNA regulates the translation of the viral polyprotein and its efficiency usually varies according to cell types. The IRES could determine the competition between HCV variants