title: Use of an in vitro model and yeast two-hybrid system to investigate the pathogenesis of hepatitis C.
abstract: Fifteen years after the discovery of hepatitis C virus (HCV) in 1989, much remains to be learnt about the cell biology of this virus. Using the serum from a patient containing HCV RNA in high titer as a source, a Singapore strain of genotype 1b was recovered and characterized. This full-length HCV genome was then constructed into a tetracycline-inducible vector using the pSTAR plasmid. Transfection of hepatoma cell lines with this HCV genome under tetracycline induction indicated that chemokines (RANTES and monocyte chemoattractant protein-1) were upregulated, possibly contributing to the induction of immune responses. Using the yeast two-hybrid system to discover protein-protein interactions, nonstructural region NS3 was found to interact with itself, forming a dimer that increased helicase activity but was not essential for its activity, thereby disqualifying it as a suitable target of drug actions. The significance of the interaction between core and NS5A is unclear, and the cleavage of NS5A is related to the development of apoptosis. However, the interaction of p68 and NS56B appears to be important because the knockdown of p68 reduced the viral replication. Finally, a new cell model using chimeric CD81 linked to the cytoplasmic domain of either a low-density lipoprotein receptor or a transferrin receptor led to productive infection of HCV that had been recovered from infected serum. These studies allow us to examine the pathogenesis of HCV infection in more detail.
authors.affiliation: Institute of Molecular and Cell Biology, Department of Medicine, National University Hospital, 5 Lower Kent Ridge Road, 119074 Singapore, Singapore. mdclimsg@nus.edu.sg
authors.forename: Seng Gee
authors.initials: SG
authors.lastname: Lim
authors.forename: Yee Joo
authors.initials: YJ
authors.lastname: Tan
authors.forename: Phuay Yee
authors.initials: PY
authors.lastname: Goh
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.forename: Wan Jin
authors.initials: WJ
authors.lastname: Hong
mesh_terms: D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D018932:Chemokine CCL2 / Q000378:metabolism; D018946:Chemokine CCL5 / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D016679:Genome, Viral; D016174:Hepacivirus / Q000378:metabolism / Q000502:physiology*; D006526:Hepatitis C / Q000821:virology*; D022781:Hepatocytes / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism; D018110:Receptors, Lipoprotein / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D060225:Tetraspanin 28; D014162:Transfection / Q000379:methods; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D015703:Antigens, CD; D018932:Chemokine CCL2; D018946:Chemokine CCL5; D053487:DEAD-box RNA Helicases; D011494:Protein Kinases; D020365:RNA Helicases; D018110:Receptors, Lipoprotein; D011990:Receptors, Transferrin; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D060225:Tetraspanin 28; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase