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Presence of hepatitis B virus precore 1896 tag mutant in the earlier stage of HBV infection
268A 645
AASLD
PRESENCE OF HEPATITIS B VIRUS PRECORE 1896 TAG MUTANT IN THE EARLIER STAGE OF HBV INFECTION H Kawai. T Yamashita. S Kaneko. K Kobavashi and *M Kinoshita First Dept. of Internal Medicine, Kanazawa University, Japan and *Diagnostic Section, Otsuka Pharmaceutical Inc., Japan
ABSTRACTS
646
under20120-29130-39140-49
PHYLLANTHUS AMARUS INTERFERES WITH TRANSCRIPTIONAL
REGULATION OF HBV ENHANCER I BY NUCLEAR TRANSCRIPTION FACTORS. M Ott.= SP Thva~,araian and S Gunta. Liver Research Center, Albert Einstein College of Medicine, B~;onx, NY, and Department of Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, India. We recently demonstrated that P. amarus plant downregulates HBVenhancer I (HBEn). To analyze mechanisms underlying this inhibitory effect, we measured HBEn activity in Huh-7 cells overexpressing transcription factors (TF). Plasmids expressing TFs HNFIII, HNF'3~, HNF311, C/EBPu or C/EBPf~ and the plasmid pHBEn/HBsP-luc containing the HBEn and HBV preSl promoter driving a luciferase gene were cotransfected via liposomes. All TF plasmids were driven by a CMV promoter except for HNFIfI, which was driven by SRu promoter. Measurement of luc activity in cell lysates 48 hrs after transfection of pHBEn/HBsP, luc showed 1.1.+0.3 x l0 s light units/mg protein (100%). The pHBEn/HBsPluc p l a s m i d was m a x i m a l l y u p r e g u l a t e d by HNF3t~ (luc activity, 323%+51% of controls, p<.001), followed by C/EBPt~ (314%+__33%, p < .001), C/EBP0 (260%+00%, p < .001), HNF38 (158%+__14%, p= .003), and least by HNF 18 (108%+9%, p=N.S.). P. amarus in doses of 100 #g/ml or 200/zg/ml inhibited pHBEn/HBsP-lue expression to 37%+16% or 25%+6% of controls, respectively, p < .001, but had no effect on luc expression in a plasmid under the SV40 enhancer/promoter. P. amarus significantly inhthited pHBEn/HBsP-Iuc expression in cells cotransfeeted with C/EBPa to 47%+ 10% (100/zg/ml) or 20%+4% (200/.tg/ml), and in cells cotransfected w~'h C/EBP0 to 46%+7% (100 #g/ml) or 34%+4% (200 #g/ml) of controls. In contrast, P. amarus did not downregulate luc activity upon cotransfection with either HNF3t~ or HNF3fl. As P. amaras could potentially suppress TF-binding to specific DNA domains, activate cellular pruteins competing with TF-binding, or alter TF mRNA processing, we used gel shift assays to analyze influence of P. a m a r u s on DNA/TF binding. P. amarus dose-dependently inhibited C/EBP-binding to a 32P-C/EBP consensus oligonucleotide in presence of a hepatocyte nuclear extract. CONCLUSIONS: P. amarus inhibits HSEn activity, selectively interferes with TF-induced upregulation of HBEn and at least in case of C/EBPs, interrupts TF-binding to specific DNA domains. These activities provide mechanistic insights into the anti-HBV effect of P. amarus and should facilitate purification of the active principle/s.
TUPAIA BELANGERh A NOVEL SMALL ANIMAL MODEL OF HEPATITIS B VIRUS INFECTION. E Waiter. R Keist. B NiederSst. I Pult, and HE Blum. Department of Medicine, University Hospital Z0rich, Rfimistrasse 100, CH-8091 Z0rich, Switzerland. Backuround: Hepatitis B virus (HBV) naturally infects only humans and experimentally chimpanzees. While many molecular and clinical aspects of HBV infection have been defined, a small animal model for the study of human HBV infection has not been available to date. Based on the close phylogenetic relationship between tree shrews and primates, we systematically analyzed the tree shrew species tupaia belangeri as an animal model for the study of HBV infection in vitro and in vivo. Methods: Tupaias were bred and maintained at the institutionalanimal facilities. Primary tupaia hepatocyte preparation and transfection analyses were performed by standard procedures. Primary tupaia hepatocytes were infected 1-2 days after preparation by overnight incubation with HBV DNA positive human serum in the presence of 4% polyethyleneglycol (PEG). For in vivo infection, tupaias were inoculated intraperitoneally with 50 h:l HBV DNA positive serum 4 days after birth. Results: Primary hepatocytes isolated from livers of the tree shrew species tupaia belangeri can be reproducibly infected with HBV./n vitro infection results in viral replication as well as synthesis of viral RNA transcripts in hepatocytes and secretion of the viral antigens hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers. Similar to acute selflimited hepatitis B in humans, HBsAg is rapidly cleared from serum, followed by seroconversion to antibodies to HBeAg (anti-HBe) and to HBsAg (anti-HBs), respectively. Conclusions: We describe a novel small animal model for the study of hepatitis B virus (HBV) infection in vitro and in vivo in tupaia belangeri. This model should permit the experimental analysis of various molecular and clinical aspects of HBV infection, including the significance of HBV quasispecies, as well as the systematic evaluation of various antivirel strategies.
Emergence of hepatitis B precore 1896 TAG mutant (TAG-mutant) has been considered major evolutionary adaptive change during late stage of HBV infection from the results of standard PeR assays. The PeR assays, however, were not sensitive enough to detect minor population of mutants. In order to elucidate the significance of TAG-mutant, we exploit highly sensitive and quantitative PeR based direct detection system for TAG-mutant in the earlier phase of infection. Methods 67 patients with HBsAg were studied: 40 were HBeAg positive, 26 were HBeAb positive. All patients except 12 with HBeAb had elevated ALT levels. 34 were serially determined for TAG-mutant for up to 9 years (mean 2.7yr). TAG-mutant specific PeR detection was done with mutation specific sense primer. Specificity of the assay was determined by sequence analysis. Quantitative measurement was done using competitive PeR assay. Results At the point of diagnosis, 48/67 (72%) were positive for TAG-mutant, and their viral titers were Ix103-3x109 co fies/ml (md; median of le :7.4). Age 50-59 I over60 TAG3/3 9/11 10/13 11/14 12/18 3/8 mutant (100%) (82%) (77%) (79%) (67%) (38%) ns 33/41 (80%) rate 15/26 (58%) )<0.05 Titer lxl0~-3xl0~(md 7.0) 2x10<2x10~(7.8) ns HBV status HBeAg positve HBeAb positive Serum ALT abnormal abnormal normal 7/12(58%) 7/14(50%) ns TAG-mutant 34/40(85%) rate 14/26(54%) p<0.01 (rod 6.8) I (md 4.5) ns Titer lxl0~-3xl0'(md 7.7) lxl0~,l~l&(md 6.4) p<0.01 Serial determination of TAG-mutant revealed reduction of the viral titer in 12/ 34 cases (34%), disappearance of TAG-mutant in 9 (26%) and de novo appearance in 5 (15%). Conclusion Highly sensitive direct TAG-mutant detection assay showed that TAG-mutant exsists with high viral copies in earlier stage of HBV infection with HBeAg. This suggests the possibility that this precore 1896 TAG mutant mac be a preserved inherent variant of HBV.
647
HEPATOLOGY O c t o b e r 1995
648
I N H I B I T I O N O F V I R A L R E P L I C A T I O N BY MUTANTS O F T H E DUCK H E P A T I T I S B VIRUS C O R E P R O T E I N . F yon Weizs~icker. S Wieland and HE Blum. Department of Medicine, University Hospital Freiburg, Germany Background. Protein variants that suppress the activity of their normal counterparts are termed "transdominant negative (DN) mutants". When derived from viral proteins, such mutants can display antiviral activity. The hepadnaviral core protein is central to the viral life cycle and thus represents an attractive target for DN mutant (Sacglioni et al., 1994). Here, we describe the generation and characterization of dominant negative duck hepatitis B virus (DHBV) core protein variants. Methods. PeR-amplified fragments from the bacterial lac Z gene expressing up to 282 amino acids were added either to the N- or C terminus of DHBV core. Also, C-terminal extensions were generated by fusing DHBV core protein with either the small surface protein or variants of the DHBV polymerase. Each mutant was tested for its potential antiviral activity by co-transfection with a replication competent DHBV construct into the avian hepatoma cell line LMH. Results. C-terminal, but not N-terminal extensions of the DHBV core protein efficiently inhibited DHBV replication. Two of the Cterminally extended core proteins suppressed viral replication by up to 90 %. Antiviral activity was shown to be species specific and independent from the amino acid sequence added. Western Blot analysis revealed that the antiviral effect was indeed due t o posttranslational interference with viral replication. Conclusions. Our findings demonstrate that the concept of inhibiting viral replication by modified core proteins is universally applicable among hepadnaviruses and provide new information about structural and sequence requirements for the design of DN core mutants. The DHBV model should be well suited to test the antiviral potential of this new class of antiviral agents in vivo.
AASLD
PRESENCE OF HEPATITIS B VIRUS PRECORE 1896 TAG MUTANT IN THE EARLIER STAGE OF HBV INFECTION H Kawai. T Yamashita. S Kaneko. K Kobavashi and *M Kinoshita First Dept. of Internal Medicine, Kanazawa University, Japan and *Diagnostic Section, Otsuka Pharmaceutical Inc., Japan
ABSTRACTS
646
under20120-29130-39140-49
PHYLLANTHUS AMARUS INTERFERES WITH TRANSCRIPTIONAL
REGULATION OF HBV ENHANCER I BY NUCLEAR TRANSCRIPTION FACTORS. M Ott.= SP Thva~,araian and S Gunta. Liver Research Center, Albert Einstein College of Medicine, B~;onx, NY, and Department of Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, India. We recently demonstrated that P. amarus plant downregulates HBVenhancer I (HBEn). To analyze mechanisms underlying this inhibitory effect, we measured HBEn activity in Huh-7 cells overexpressing transcription factors (TF). Plasmids expressing TFs HNFIII, HNF'3~, HNF311, C/EBPu or C/EBPf~ and the plasmid pHBEn/HBsP-luc containing the HBEn and HBV preSl promoter driving a luciferase gene were cotransfected via liposomes. All TF plasmids were driven by a CMV promoter except for HNFIfI, which was driven by SRu promoter. Measurement of luc activity in cell lysates 48 hrs after transfection of pHBEn/HBsP, luc showed 1.1.+0.3 x l0 s light units/mg protein (100%). The pHBEn/HBsPluc p l a s m i d was m a x i m a l l y u p r e g u l a t e d by HNF3t~ (luc activity, 323%+51% of controls, p<.001), followed by C/EBPt~ (314%+__33%, p < .001), C/EBP0 (260%+00%, p < .001), HNF38 (158%+__14%, p= .003), and least by HNF 18 (108%+9%, p=N.S.). P. amarus in doses of 100 #g/ml or 200/zg/ml inhibited pHBEn/HBsP-lue expression to 37%+16% or 25%+6% of controls, respectively, p < .001, but had no effect on luc expression in a plasmid under the SV40 enhancer/promoter. P. amarus significantly inhthited pHBEn/HBsP-Iuc expression in cells cotransfeeted with C/EBPa to 47%+ 10% (100/zg/ml) or 20%+4% (200/.tg/ml), and in cells cotransfected w~'h C/EBP0 to 46%+7% (100 #g/ml) or 34%+4% (200 #g/ml) of controls. In contrast, P. amarus did not downregulate luc activity upon cotransfection with either HNF3t~ or HNF3fl. As P. amaras could potentially suppress TF-binding to specific DNA domains, activate cellular pruteins competing with TF-binding, or alter TF mRNA processing, we used gel shift assays to analyze influence of P. a m a r u s on DNA/TF binding. P. amarus dose-dependently inhibited C/EBP-binding to a 32P-C/EBP consensus oligonucleotide in presence of a hepatocyte nuclear extract. CONCLUSIONS: P. amarus inhibits HSEn activity, selectively interferes with TF-induced upregulation of HBEn and at least in case of C/EBPs, interrupts TF-binding to specific DNA domains. These activities provide mechanistic insights into the anti-HBV effect of P. amarus and should facilitate purification of the active principle/s.
TUPAIA BELANGERh A NOVEL SMALL ANIMAL MODEL OF HEPATITIS B VIRUS INFECTION. E Waiter. R Keist. B NiederSst. I Pult, and HE Blum. Department of Medicine, University Hospital Z0rich, Rfimistrasse 100, CH-8091 Z0rich, Switzerland. Backuround: Hepatitis B virus (HBV) naturally infects only humans and experimentally chimpanzees. While many molecular and clinical aspects of HBV infection have been defined, a small animal model for the study of human HBV infection has not been available to date. Based on the close phylogenetic relationship between tree shrews and primates, we systematically analyzed the tree shrew species tupaia belangeri as an animal model for the study of HBV infection in vitro and in vivo. Methods: Tupaias were bred and maintained at the institutionalanimal facilities. Primary tupaia hepatocyte preparation and transfection analyses were performed by standard procedures. Primary tupaia hepatocytes were infected 1-2 days after preparation by overnight incubation with HBV DNA positive human serum in the presence of 4% polyethyleneglycol (PEG). For in vivo infection, tupaias were inoculated intraperitoneally with 50 h:l HBV DNA positive serum 4 days after birth. Results: Primary hepatocytes isolated from livers of the tree shrew species tupaia belangeri can be reproducibly infected with HBV./n vitro infection results in viral replication as well as synthesis of viral RNA transcripts in hepatocytes and secretion of the viral antigens hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers. Similar to acute selflimited hepatitis B in humans, HBsAg is rapidly cleared from serum, followed by seroconversion to antibodies to HBeAg (anti-HBe) and to HBsAg (anti-HBs), respectively. Conclusions: We describe a novel small animal model for the study of hepatitis B virus (HBV) infection in vitro and in vivo in tupaia belangeri. This model should permit the experimental analysis of various molecular and clinical aspects of HBV infection, including the significance of HBV quasispecies, as well as the systematic evaluation of various antivirel strategies.
Emergence of hepatitis B precore 1896 TAG mutant (TAG-mutant) has been considered major evolutionary adaptive change during late stage of HBV infection from the results of standard PeR assays. The PeR assays, however, were not sensitive enough to detect minor population of mutants. In order to elucidate the significance of TAG-mutant, we exploit highly sensitive and quantitative PeR based direct detection system for TAG-mutant in the earlier phase of infection. Methods 67 patients with HBsAg were studied: 40 were HBeAg positive, 26 were HBeAb positive. All patients except 12 with HBeAb had elevated ALT levels. 34 were serially determined for TAG-mutant for up to 9 years (mean 2.7yr). TAG-mutant specific PeR detection was done with mutation specific sense primer. Specificity of the assay was determined by sequence analysis. Quantitative measurement was done using competitive PeR assay. Results At the point of diagnosis, 48/67 (72%) were positive for TAG-mutant, and their viral titers were Ix103-3x109 co fies/ml (md; median of le :7.4). Age 50-59 I over60 TAG3/3 9/11 10/13 11/14 12/18 3/8 mutant (100%) (82%) (77%) (79%) (67%) (38%) ns 33/41 (80%) rate 15/26 (58%) )<0.05 Titer lxl0~-3xl0~(md 7.0) 2x10<2x10~(7.8) ns HBV status HBeAg positve HBeAb positive Serum ALT abnormal abnormal normal 7/12(58%) 7/14(50%) ns TAG-mutant 34/40(85%) rate 14/26(54%) p<0.01 (rod 6.8) I (md 4.5) ns Titer lxl0~-3xl0'(md 7.7) lxl0~,l~l&(md 6.4) p<0.01 Serial determination of TAG-mutant revealed reduction of the viral titer in 12/ 34 cases (34%), disappearance of TAG-mutant in 9 (26%) and de novo appearance in 5 (15%). Conclusion Highly sensitive direct TAG-mutant detection assay showed that TAG-mutant exsists with high viral copies in earlier stage of HBV infection with HBeAg. This suggests the possibility that this precore 1896 TAG mutant mac be a preserved inherent variant of HBV.
647
HEPATOLOGY O c t o b e r 1995
648
I N H I B I T I O N O F V I R A L R E P L I C A T I O N BY MUTANTS O F T H E DUCK H E P A T I T I S B VIRUS C O R E P R O T E I N . F yon Weizs~icker. S Wieland and HE Blum. Department of Medicine, University Hospital Freiburg, Germany Background. Protein variants that suppress the activity of their normal counterparts are termed "transdominant negative (DN) mutants". When derived from viral proteins, such mutants can display antiviral activity. The hepadnaviral core protein is central to the viral life cycle and thus represents an attractive target for DN mutant (Sacglioni et al., 1994). Here, we describe the generation and characterization of dominant negative duck hepatitis B virus (DHBV) core protein variants. Methods. PeR-amplified fragments from the bacterial lac Z gene expressing up to 282 amino acids were added either to the N- or C terminus of DHBV core. Also, C-terminal extensions were generated by fusing DHBV core protein with either the small surface protein or variants of the DHBV polymerase. Each mutant was tested for its potential antiviral activity by co-transfection with a replication competent DHBV construct into the avian hepatoma cell line LMH. Results. C-terminal, but not N-terminal extensions of the DHBV core protein efficiently inhibited DHBV replication. Two of the Cterminally extended core proteins suppressed viral replication by up to 90 %. Antiviral activity was shown to be species specific and independent from the amino acid sequence added. Western Blot analysis revealed that the antiviral effect was indeed due t o posttranslational interference with viral replication. Conclusions. Our findings demonstrate that the concept of inhibiting viral replication by modified core proteins is universally applicable among hepadnaviruses and provide new information about structural and sequence requirements for the design of DN core mutants. The DHBV model should be well suited to test the antiviral potential of this new class of antiviral agents in vivo.