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Specific Targeting of Hepatitis C Virus Core Protein by an Intracellular Single-Chain Antibody of Human Origin Juliane Karthe, Kathi Tessmann,et al. HEPATOLOGY, Vol. 48, No. 3, 2008 -----Presented by Gan Weiqiang Background Hepatitis C Hepatitis C virus (HCV): discovered in 1989, infects approximately 200 million people worldwide. Persistent infection:chronic hepatitis, hepatic steatosis, cirrhosis, HCC. HCV:small, enveloped RNA virus, Hepacivirus genus of the Flaviviridae family. Experimental models of hepatitis C virus infection, replication, and pathogenesis. Hepatology 2001;33:489–95. Background HCV genome structure Hepatitis C virus proteins World J Gastroenterol 2007 May 7; 13(17): 2406-2415 Background HCV protein and its function Hepatitis C virus proteins World J Gastroenterol 2007 May 7; 13(17): 2406-2415 Background Core Protein Description:Structural Protein, part of the viral nucleocapsid Position:N-terminal of the polyprotein Formation: cleavage at ER by signal peptidases Background Structure N-terminus: Hydrophilic, highly basic and contains RNA and DNA binding domains, nuclear localization signals. C-terminus: Hydrophobic, mediates anchorage to the ER. Background Form Longer form (p21):residues 1-191,localizes to the cytoplasm related to ER membrane. Shorter form (p19): residues 1-173,derived from p21. Translocated to the nucleus. Background Function Steatosis cell growth carcinogenesis Core Protein apoptosis signaling Background Function Hepatic steatosis: Function Chronic hepatitis C Intrahepatic core protein Hepatic steatosis In human hepatoma cells: upregulate transcription of most genes involved in fat/lipid metabolism. Background Function CORE PROTEIN Interact with and maintain an activated form of (PKR) the RNAdependent protein kinase mitogen-activated protein kinase /extracellular signalrelated kinase Activate MAPK/ERK pathway, Wnt1 signaling HCC Background Challenges Lack of a preventive vaccine Lack of effective antiviral therapies New solution in need! Background scFv---single-chain variable fragments Background Advantages of scFv Small Molecular Weight,high permiability! Low immunogenicity----can be used repeatedly! With easy gene process----can be obtain in large quantity! Background Fuction of scFv Folding Interaction alter target Protein Modification Subcellular localization Potential therapeutics against cancer and HIV Background How do we get it? Phage Display Technology Background Why do we use scFv? Core protein:most conserved HCV protein ---have a broad effect on different genotypes Viral life cycle and pathogenesis---1.Abrogate core particle assembly and RNA packaging 2.Affect its direct effect on cell proliferation and lipid metabolism Background Research purposes Develop a human antibody fragment scFv against HCV core protein ; Investigate its intracellular distribution; Find out its association with the core protein; Look into its impact on cell proliferation triggered by the core protein. Background Research Method Technology Routine Extract Bone marrow Human scFv Library Affinity Selection EIA Transfect Huh7 cells Construct plasmids of scFV42C and Core Protein Map the Epitope Recognized by scFv42C Immunofluorescence Confocal Laser Scanning and FRET Immunoblotting DNA Sequenc ing Expression and Purificationof scFv42C Cell Proliferation Assay. Method Extract Bone marrow Patients: Serologically documented HCV infection (HCV antibody by third-generation enzyme-linked immunoassay) Serum HCV-RNA by reverse transcription polymerase chain reaction(RT-PCR) Method Human scFv Library 1. PCR human VH and VL fragments from bone marrow aspirate extracts 2. Cloned into the phagemid vector pAK100.25 3. Expression of an scFv as a gene III fusion protein on the surface of filamentous phages Method Affinity Selection EIA Extract Bone marrow Core protein Phage Display Technology Method Map the Epitope Recognized by scFv42C. Identify peptide sequences recognized by the scFv42C antibody: 1.The Ph.D.-7 phage display library peptides kit containing linear peptides(randomly presenting 7-12 amino acids) 2.Selected clones were sequenced and the amino acid sequence of the presented peptides was deduced to determine the consensus binding motif. Method Molecular cloning 1. scFv42C-YFP and scFvIR-YFP: cDNA encoding scFv42C and scFvIR (irrelevant scFv) antibody fragments were cloned into pEYFP-N1 with its C-terminus fused to enhanced yellow fluorescence protein(YFP) 2.pEF/sc42:scFv42C cDNA was cloned into EF/myc/cyto vector Method Molecular cloning 3.pZeo/core: cDNA encoding the full-length core protein (aa 1-191 of 1b genotype)was cloned into pcDNA3.1/Zeo(+) vector 4. CFP-C191:cDNA encoding the full-length core protein (aa 1-191 of 1b genotype)was subcloned into pRc/CMV and pECFP-C1 vector with Nterminal fusion of the enhanced cyan fluorescence protein (CFP) Method Transfection Experiments CFP-C191/Core-pRc/CMV scFv42C-YFP/scFvIR-YFP Transfect Huh7 for pZeo/core and pEF/sc42 Transfect Huh7 for colocalization assay Western blot JFH1(replicon RNA) and pEF/sc42 Transfect Huh7.5 for Western and Northern blot Method Colocalization Experiments Immunofluorescence Confocal Laser Scanning Microscope Fluorescence Resonance Energy Transfer Technique(FRET). Method Cell Proliferation Assay Cell proliferation:day 1 or 2 after transfection Add tetrazolium to medium Measure absorbence at 450 nm after 1-4 hours Method Results Identification of High-Affinity Single-Chain Antibodies Against HCV Core Protein. Results Amino Acid Sequence Results Purification of scFv42C Figure 3 Results Verification The purified antibody could recognize the core protein (aa 1-115) in Western blot, albeit the signal was much weaker than that of mouse anti-core antibody (Fig.3B). Figure 3 Results Mapping of the scFv42C Binding Motif on the Core Protein. Phage Display Peptide Library scFv42C Phage Display Technology Results Mapping of the scFv42C Binding Motif on the Core Protein. Results Intracellular Colocalization of scFv42C with HCV Core Protein. Confocal laser scanning microscope was used to investigate intracellular interaction between scFv42C and core protein. To facilitate their detection,CFP was attached to the N-terminus of core protein(CFP-191),whereas YFP was tagged to the C-terminus of scFv42C (scFv42C-YFP) or scFvIR (scFvIR-YFP). Results Localization of Core Protein and scFv42c individually Figure 5 Results Coexpression of scFv42C with the core protein Figure 5 Coexpression of scFv42C with the core protein shifted the scFv42C expression pattern to globular-like, which colocalized with HCV core. Results FRET Fluorescence Resonance Energy Transfer Technique Core protein scFv-IR Core protein scFv-42C Results FRET Analysis Results scFv42C’s reduce Core protein The scFv42C Antibody Reduced Core Protein Level but not Viral Replication per se in Transient Transfection. Results Cotransfect with core Results Cotransfect with HCV Results Transfect with HCV Results Effect of scFv42C on cell proliferation Results Discussion Advantages of scFv over intact antibody Smaller size Ease of production Improved tissue penetration Discussion Applications In the study of HIV 1.scFvs targeting the tat or VIF protein: Inhibite HIV replication; 2.scFvs against the integrase: Confer cellular resistance to HIV infection Discussion Applications As for cancer therapy 1.scFvs targeting vascular endothelial growth factor receptor 2: Reduce the malignant potential of melanoma cells 2.scFvs recognizing lung cancer–associated common antigens: Reduce the growth of lung cancer cells Discussion The case of HCV HCV causes serious diseases: steatosis HCC fibrosis cirrhosis Single-chain antibodies may prove useful in the therapy of chronic HCV infection. Discussion The case of HCV scFvs against NS3 (helicase) and NS5B (RNAdependent RNA polymerase): Effective in inhibiting their enzymatic activities both in vitro and in transfected cells. scFvs represent potential candidates for intracellular immunization against HCV infection. Discussion scFv against core protein Core protein:a major viral factor contributing to HCV pathogenesis. Targeting the viral core protein may alleviate HCV-related pathological changes. Discussion Verification High affinity scFv against core protein Features:recognize motif PXPLXG Interact with core protein:deplete core protein and its pathogenesis Interrupt viral life cycle? Discussion Interrupt viral life cycle? In this study: scFv42C antibody did not directly affect HCV replication in transient transfection. Reason: Core protein: 1.Not required for viral genome replication 2.Necessary for virus particle assembly. Discussion Interrupt viral life cycle? Mechanism: Interfere with nucleocapsid formation or RNA encapsidation. Unpublished data: scFv42C antibody reduce viral particle formation. Discussion scFv HCV