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


CONCLUSIONS
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.
Feng
De-Yun
DY
Department of Pathology, College of Basic Medicine, Central South University, Changsha 410078, Hunan Province, China. dyfeng743@sohu.com
Sun
Yi
Y
Cheng
Rui-Xue
RX
Ouyang
Xiao-Ming
XM
Zheng
Hui
H
D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology / Q000821:virology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010957:Plasmids; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020929:Mitogen-Activated Protein Kinase Kinases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases