Effect of hepatitis C virus nonstructural protein NS3 on proliferation and MAPK phosphorylation of normal hepatocyte line

Abstract

Aim: To study the effect of hepatitis C virus nonstructural region 3 (HCV NS3) protein on proliferation and transformation of normal human liver cell line.

Methods: QSG7701 cells were transfected with pRcHCNS3-5', pRcHCNS3-3' and pRcCMV using lipofectamine transfecting technique and selected with G418 method. Expression of HCV NS3 protein was determined by immunohistochemistry. Biologic characteristics of transfected cells were evaluated by population doubling time and soft agar assays. Activation of MAPK was analyzed using Western blot with phosphospecific monoclonal antibody against dually phosphorylated MAPK.

Results: QSG7701 cells transfected with pRcHCNS3-5' showed strong intracellular expression of HCVNS3 protein, and the positive signal was localized in cytoplasm. The expressing strength of HCVNS3 protein in pRcHCNS3-3'-transfected cells was weaker than that in pRcHCNS3-5'-transfected cells. The population doubling time in the transfected cells with pRcHCNS3-5' (12 h) was much shorter than those with pRcHCNS3-3', pRcCMV and normal cells (24, 26, 28 h, respectively) (P<0.01). The transfected cells with pRcHCNS3-5' showed much more anchorage independent colonies than that in those with pRcHCNS3-3' and pRcCMV (P<0.01). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcHCNS3-3', pRcCMV and controls were 33%, 1.33%, 1.46%, 1.11% respectively. The level of phosphorylated MAPK in the cells with pRcHCNS3-5' was much higher than that in those with pRcHCNS3-3'and pRcCMV and normal cells (P<0.01).

Conclusion: The results suggest that (1) QSG7701 cells are a better human liver cell line for investigating the pathogenesis of HCV NS3 protein. (2) 5' region of the HCV genome segment encoding HCV NS3 is involved in cell growth and cell phenotype. (3) HCV NS3 N-terminal peptide may up-regulate the activation of MAPK, but not affect the expression of MAPK.

Figure 1

Agarose gel electrophoresis analysis of pRcHCN3-3’ and pRcHCN3-5’ digested with XbaI. Lane M: DNA marker (λDNA/Hind III). Lanes 1 and 2: pRcHCNS3-5’ plasmid, 886-bp fragment. Lanes 3 and 4: pRcHCNS3-3’ plasmid, 1 031-bp fragment.

Figure 2

Clones of QSG7701 cell transfected with pRcHCNS3-5’ and pRcHCN3-3’ plasmid in soft agar. A: pRcHCNS3-5’ transfected group. B: pRcHCNS3-3’ transfected group (×100).

Figure 3

Identification of expression plasmid of HCNS3 protein in QSG7701 cells. Lane 1: pRcHCNS3-5’; lane 2: pRcCMV; lane 3: untransfected QSG7701 cells; lane 4: blank control.

Figure 4

Expression of HCV NS3 protein in QSG77 cell transfected with pRcHCNS3-5’ plasmid. The positive products were localized in cytoplasm. SP ×400.

Figure 5

Growth curve of four kinds of cells.

Figure 6

Western blot analysis of phosphorylated (A) and non-phosphorylated (B) MAPK in each group. Lanes 1 and 2: non-transfected group; Lanes 3 and 4: pRcCMV transfected group; Lanes 5-7: pRcHCNS3-5’ transfected group; Lanes 8-10: pRcHCNS3-3’ transfected group.