- Email: [email protected]
B TARGET GENES REPERTOIRES MODULATED BY EXOGENOUS TYPE I IFN AND BY THE ENDOGENOUS HBV-INDUCED IFN-RESPONSE IN LIVER CELLS
POSTERS 629 IMMUNE ESCAPE MUTATIONS G145R AND P120T DIFFERENTIALLY IMPACT THE REPLICATION EFFICIENCY OF LAMIVUDINE-RESISTANT HEPATITIS B VIRUS E ANTIGEN-POSITIVE AND -NEGATIVE STRAINS S. Amini-Bavil-Olyaee, C. Trautwein, F. Tacke. Medizinische Klinik III, Universit¨ atsklinikum Aachen, Aachen, Germany E-mail: [email protected] Background and Aims: Immune escape variants of the hepatitis B virus (HBV) with alterations in the HBs envelope proteins represent an emerging clinical challenge, because they can be associated with vaccine escape, HBV reactivation and failure of diagnostic tests. Recent data suggest a preferential selection of immune escape mutants in distinct peripheral blood leukocyte compartments of infected individuals. We therefore systematically analyzed the functional impact of the most prevalent immune escape variants, sG145R and sP120T, on the viral replication efficacy and antiviral drug susceptibility of common treatment-associated mutants with resistance to lamivudine (LAM) and/or HBeAg-negativity. Methods: Replication-competent HBV strains with sG145R or sP120T and LAM-resistance (rtM204I or rtL180M/rtM204V) were generated on an HBeAg-positive and on an HBeAg-negative background with precore (PC) and basal core promoter (BCP) mutants. After transient transfection in human hepatoma cells, replication was assessed by Northern Blot, Progeny-DNA, qPCR and HBV protein assays. Results: The sG145R mutation strongly reduced HBsAg levels and was able to fully restore the impaired replication of LAM-resistant HBV mutants to levels of wild-type HBV, and PC or BCP mutations further enhanced viral replication. Although the sP120T substitution also impaired HBsAg secretion, it did not enhance the replication of LAM-resistant clones. However, the concomitant occurrence of HBeAg-negativity (PC/BCP), sP120T and LAM-resistance resulted in restoration of replication to levels of wild-type HBV. In all clones with combined immune escape and LAM-resistance mutations, the nucleotide analogues adefovir and tenofovir remained effective in suppressing viral replication in vitro. Conclusions: These findings reveal the differential impact of immune escape variants on the replication and drug susceptibility of complex HBV mutants, supporting the need of close surveillance and treatment adjustment in response to the selection of distinct mutational patterns. 630 HEPATITIS B VIRUS X PROTEIN AUGMENTS HBV TRANSCRIPTION AND REPLICATION IN VIVO OF MOUSE F.-J. Huang1 , H. Tang2,3 , Q.-L. Hou1 , E.-Q. Chen2,3 , F.-J. Liu2,3 , F. He2,3 . 1 Department of Forensic Pathology, Medical School of Basic and Forensic Sciences, Sichuan University, 2 Center of Infectious Diseases, West China Hospital, Sichuan University, 3 Division of Infectious Diseases, State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China E-mail: [email protected] Background and Aims: Hepatitis B virus (HBV) X protein (HBx) is a 154 amino acids (154-aa) protein, with an N-terminal negative regulatory domain and a C-terminal transactivation or coactivation domain. The role of HBx in regulating HBV transcription and replication were investigated in HepG2 in vitro. The execution of HBx function requires the participation of host cell protein factors, whose types and levels may be different between HepG2 cell lines in vitro and liver cells in vivo. The purpose of this study was to investigate the possible role of HBx in regulating HBV transcription and replication in vivo. Methods: HBV replication mouse models were established using hydrodynamic tail vein injection of wild-type or HBx-minus HBV genome(with defective X gene), and HBx expression plasmid. After
injection, mice were sacrificed on day 3, and liver tissue was collected and total RNA and DNA were extracted. The levels of HBV mRNA in the liver were analyzed by Northern blot hybridization, and the levels of HBV DNA replication intermediates in the liver were detected by Southern blot hybridization. Results: The levels of 3.5-kb HBV RNA and HBV DNA replication intermediates of HBx-minus HBV mutants in liver were lower than that of wild-type HBV genome or HBx-minus HBV genome plus HBx expression plasmid. The absence of HBx expression caused the reduction of HBV transcription and replication and the decreases could be restored by expression of HBx in liver tissue. Conclusions: Our results showed that HBx has augmentation effects on HBV transcription and replication in vivo of mouse. This work was supported by the National Natural Science Foundation of China, No. 30570064. 631 IFN-A/B TARGET GENES REPERTOIRES MODULATED BY EXOGENOUS TYPE I IFN AND BY THE ENDOGENOUS HBV-INDUCED IFN-RESPONSE IN LIVER CELLS B. Testoni1,2,3 , J. Lucifora4 , L. Belloni1,2,3 , C. Scisciani1,2 , D. Durantel4,5 , F. Zoulim4,6 , M. Levrero1,2,3 . 1 Dept of Internal Medicine and AIRC Rome Oncogenomic Center (ROC), Sapienza University of Rome, 2 Laboratory of Gene Expression, Fondazione A Cesalpino, Rome, 3 EAL U785, INSERM, Villejuif/Rome, Italy; 4 U871, INSERM, 5 IFR62, Universit´e Lyon, 6 Hospices Civils de Lyon, Hˆ otel Dieu Hospital, Lyon, France E-mail: [email protected] Background: Microarrays and ChIP-on-chip studies have greatly broadened class IFNs transcriptome to include a number of “new” target genes beyond the classical “inflammation” and “immune response” classic genes. We have designed a multiple real time PCR liquid array to trace the expression of 96 “classical” and “new” ISGs, all confirmed Stat2 direct transcriptional targets by chromatin immunoprecipitation techniques (ChIP-on-chip). It has recently been shown that recombinant HBV baculoviruses (BacHBV) initiate a complete HBV replication cycle in both HCC cell lines and in non-transformed hepatocytic HepaRG cells. IFN-beta and a number of classical interferon stimulated genes (ISGs) are up-regulated early post-infection in both HepG2 and HepaRG cells transduced by Bac-HBV. Aim: To compare the repertoires of target genes modulated by exogenous type I IFN in human liver cell lines and in human primary hepatocytes (PHH) and by the endogenous HBV-induced IFN-response. Methods: mRNAs were prepared from PHH, HepG2 and HepaRG exposed to exogenous IFN for 2, 4, 8, 24 and 48 h or infected with HBV or control baculovitus. Gene expression data were was analyzed using Spotfire® data mining tools. Results: The profile of genes activated in response to exogenous IFN in PHH and liver cell lines is similar, although differences can be found both in the kinetics and the strenght of the responses. On the opposite, striking differences can be observed in the repertoire of repressed genes which overlap only minimally. In general, HCC cell lines seem to be more “refractory” to IFN-mediated trancriptional repression. The analysis of the 96 ISGs expression profile in baculoHBV infected cells identify 4 sub-clusters. As expected, HepaRG with RNAi for IFNRA1 show no activation of most ISGs with the exception of PRAME, CXCL11, OAS1 and MX2, which could be regulated by an additional regulatory pathway. Kinetics of ISGs activation and repression differ between cell lines. In HepaRG and PHH cells but not in HepG2 there is a selective activation of “cell-cycle arrest” and “apoptosis” genes. Conclusions: We confirm that HBV modulates the type I IFN transcriptome in human hepatocytic cell lines. The repertoire of repressed genes differentiates HBV-induced and exogenous IFN responses.
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injection, mice were sacrificed on day 3, and liver tissue was collected and total RNA and DNA were extracted. The levels of HBV mRNA in the liver were analyzed by Northern blot hybridization, and the levels of HBV DNA replication intermediates in the liver were detected by Southern blot hybridization. Results: The levels of 3.5-kb HBV RNA and HBV DNA replication intermediates of HBx-minus HBV mutants in liver were lower than that of wild-type HBV genome or HBx-minus HBV genome plus HBx expression plasmid. The absence of HBx expression caused the reduction of HBV transcription and replication and the decreases could be restored by expression of HBx in liver tissue. Conclusions: Our results showed that HBx has augmentation effects on HBV transcription and replication in vivo of mouse. This work was supported by the National Natural Science Foundation of China, No. 30570064. 631 IFN-A/B TARGET GENES REPERTOIRES MODULATED BY EXOGENOUS TYPE I IFN AND BY THE ENDOGENOUS HBV-INDUCED IFN-RESPONSE IN LIVER CELLS B. Testoni1,2,3 , J. Lucifora4 , L. Belloni1,2,3 , C. Scisciani1,2 , D. Durantel4,5 , F. Zoulim4,6 , M. Levrero1,2,3 . 1 Dept of Internal Medicine and AIRC Rome Oncogenomic Center (ROC), Sapienza University of Rome, 2 Laboratory of Gene Expression, Fondazione A Cesalpino, Rome, 3 EAL U785, INSERM, Villejuif/Rome, Italy; 4 U871, INSERM, 5 IFR62, Universit´e Lyon, 6 Hospices Civils de Lyon, Hˆ otel Dieu Hospital, Lyon, France E-mail: [email protected] Background: Microarrays and ChIP-on-chip studies have greatly broadened class IFNs transcriptome to include a number of “new” target genes beyond the classical “inflammation” and “immune response” classic genes. We have designed a multiple real time PCR liquid array to trace the expression of 96 “classical” and “new” ISGs, all confirmed Stat2 direct transcriptional targets by chromatin immunoprecipitation techniques (ChIP-on-chip). It has recently been shown that recombinant HBV baculoviruses (BacHBV) initiate a complete HBV replication cycle in both HCC cell lines and in non-transformed hepatocytic HepaRG cells. IFN-beta and a number of classical interferon stimulated genes (ISGs) are up-regulated early post-infection in both HepG2 and HepaRG cells transduced by Bac-HBV. Aim: To compare the repertoires of target genes modulated by exogenous type I IFN in human liver cell lines and in human primary hepatocytes (PHH) and by the endogenous HBV-induced IFN-response. Methods: mRNAs were prepared from PHH, HepG2 and HepaRG exposed to exogenous IFN for 2, 4, 8, 24 and 48 h or infected with HBV or control baculovitus. Gene expression data were was analyzed using Spotfire® data mining tools. Results: The profile of genes activated in response to exogenous IFN in PHH and liver cell lines is similar, although differences can be found both in the kinetics and the strenght of the responses. On the opposite, striking differences can be observed in the repertoire of repressed genes which overlap only minimally. In general, HCC cell lines seem to be more “refractory” to IFN-mediated trancriptional repression. The analysis of the 96 ISGs expression profile in baculoHBV infected cells identify 4 sub-clusters. As expected, HepaRG with RNAi for IFNRA1 show no activation of most ISGs with the exception of PRAME, CXCL11, OAS1 and MX2, which could be regulated by an additional regulatory pathway. Kinetics of ISGs activation and repression differ between cell lines. In HepaRG and PHH cells but not in HepG2 there is a selective activation of “cell-cycle arrest” and “apoptosis” genes. Conclusions: We confirm that HBV modulates the type I IFN transcriptome in human hepatocytic cell lines. The repertoire of repressed genes differentiates HBV-induced and exogenous IFN responses.
Journal of Hepatology 2010 vol. 52 | S183–S317
S247