Transactivating effect of hepatitis C virus core protein: A suppression subtractive hybridization study

AIM:To investigate the transactivating effect of hepatitis Cvirus(HCV)core protein and to screen genes transactivatedby HCV core protein.METHODS:pcDNA3.1(-)-core containing full-length HCVcore gene was constructed by insertion of HCV core geneinto EcoRI/BamHI site.HepG2 cells were cotransfected withpcDNA3.1(-)-core and pSV-lacZ.After 48 h,cells werecollected and detected for the expression of β-gal by anenzyme-linked immunosorbent assay(ELISA)kit.HepG2 cellswere transiently transfected with pcDNA3.1(-)-core usingLipofectamine reagent.Cells were collected and total mRNAwas isolated.A subtracted cDNA library was generated andconstructed into a pGEM-Teasy vector.The library wasamplified with E.coli strain JM109.The cDNAs weresequenced and analyzed in GenBank with BLAST search afterpolymerase chain reaction(PCR).RESULTS:The core mRNA and protein could be detected inHepG2 cell lysate which was transfected by the pcDNA3.1(-)-core.The activity of β-galactosidase in HepG2 cells transfectedby the pcDNA3.1(-)-core was 5.4 times higher than that ofHepG2 cells transfected by control plasmid.The subtractivelibrary of genes transactivated by HCV core protein wasconstructed successfully.The amplified library contained 233positive clones.Colony PCR showed that 213 clonescontained 100-1000 bp inserts.Sequence analysis wasperformed in 63 clones.Six of the sequences were unknowngenes.The full length sequences were obtained withbioinformatics method,accepted by GenBank.It wassuggested that six novel cDNA sequences might be targetgenes transactivated by HCV core protein.CONCLUSION:The core protein of HCV has transactivatingeffects on SV40 early promoter/enhancer.A total of 63 clonesfrom cDNA library were randomly chosen and sequenced.Using the BLAST program at the National Center forBiotechnology Information,six of the sequences wereunknown genes.The other 57 sequences were highly similarto known genes.