Transactivating effect of hepatitis C virus core protein: a suppression subtractive hybridization study.
OBJECTIVE
To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein.


METHODS
pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BamHI site. HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ. After 48 h, cells were collected and detected for the expression of beta-gal by an enzyme-linked immunosorbent assay (ELISA) kit. HepG2 cells were transiently transfected with pcDNA3.1(-)-core using Lipofectamine reagent. Cells were collected and total mRNA was isolated. A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector. The library was amplified with E. coli strain JM109. The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR).


RESULTS
The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core. The activity of beta-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid. The subtractive library of genes transactivated by HCV core protein was constructed successfully. The amplified library contained 233 positive clones. Colony PCR showed that 213 clones contained 100-1 000 bp inserts. Sequence analysis was performed in 63 clones. Six of the sequences were unknown genes. The full length sequences were obtained with bioinformatics method, accepted by GenBank. It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein.


CONCLUSIONS
The core protein of HCV has transactivating effects on SV40 early promoter/enhancer. A total of 63 clones from cDNA library were randomly chosen and sequenced. Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes. The other 57 sequences were highly similar to known genes.
Liu
Min
M
Department of Infectious Diseases, The First Hospital of Xi'an Jiaotong University, Shaanxi Province, China.
Liu
Yan
Y
Cheng
Jun
J
Zhang
Shu-Lin
SL
Wang
Lin
L
Shao
Qing
Q
Zhang
Jian
J
Yang
Qian
Q
D015967:Gene Expression Regulation, Viral; D015723:Gene Library; D016174:Hepacivirus / Q000235:genetics*; D018197:Hepatoblastoma; D006801:Humans; D007763:Lac Operon; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015533:Transcriptional Activation; D014758:Viral Core Proteins / Q000235:genetics*
D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus