Analysis of hepatitis C virus NS3 protein having transforming activity in mice

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In Japan, a close relation between continued infection of hepatitis C virus (HCV) and liver cell cancer is wellknown. However, its mechanism is not clear. HCV (Hepacivirus) is known to show the structure same as Flavivirus. Paying an attention to this point, we have examined the function of a NS3 domain of HCV. And we discovered that HCV-NS3 protein showed a tumor formation ability for mouse cell NIH3T3 (J.Virol.,69:3893, 1995). An interaction of the host protein such as p53 and NS3 can be speculated. We tested this and found that in the NS3 and p53 vector introduced cells, both the cell-increase ability and the ability of the tumor formation in nude mouse were significantly decreased. To identify a new host protein that could interact with HCV-NS3, we performed a yeast two-hybrid screening using a HeLa cDNA library, and finally selected two NS3-binding proteins, Sm-D1 and SRCAP. Sm-DI is a component of small nuclear ribonucleoprotein complexes, and SRCAP is a protein relating to cell transcription activity. Both in vitro and in vivo bindings of Sm-D1 and NS3 were conducted using a constructed Sm-D1 and glutathione S-transferase fusion protein expression vector and a constructed FLAG-tagged Sm-D1 expression vector, respectively, and revealed that the C-terminal region of Sm-D1 containing the GR repeats was the binding region for NS3. In addition, the expression feature of Sm-D1 was affected by co-expression of NS3. Immunostaining assay using KN73 cells demonstrated that NS3 protein, which is usually localized in the cytoplasm of cells, was detectable in the nucleus when both NS3 and the host proteins were transfected into the cells. The nuclear shift of NS3 protein was greater in co-expression with Sm-D1 than that with SRCAP. These results suggest that the host proteins, especially nuclear proteins could connect with HCV-NS3 and may correlate closely with the cell transformation mechanism of HCV-NS3.