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. 2004 Jun 15;10(12):1746-9.
doi: 10.3748/wjg.v10.i12.1746.

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

Min Liu  1 , Yan Liu, Jun Cheng, Shu-Lin Zhang, Lin Wang, Qing Shao, Jian Zhang, Qian Yang
Affiliations

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

Min Liu et al. World J Gastroenterol. .

Abstract

Aim: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Products of pcDNA3.1(-)-core PCR and restriction enzyme cleavage were electrophoresed in 1% agarose gel. Lane 1: EcoRI/BamHI cleaved; lane 2: HindIII cleaved; lane 3: prod-ucts of plasmid PCR; M: DNA marker, (15 000 bp+ 2 000 bp).
Figure 2
Figure 2
RT-PCR products were electrophoresed in 1% agar-ose gel. Lane 1: negative control; lanes 2-4: total RNA was iso-lated from pcDNA3.1(-)-core and RT-PCR was performed by three different Oligo dT; lane 5: blank control; lane 6: positive control; M: DNA marker (2 000 bp).
Figure 3
Figure 3
Comparison of A values among transfection groups. 1. Negative control (PBS); 2. pcDNA3.1(-)-core; 3. Blank plasmid; 4. Positive control.
Figure 4
Figure 4
Agarose gel electrophoresis of PCR products of some clones (30-45). M: DNA marker (2 000 bp).
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