OBJECTIVETo study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.

METHODSPersistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins.

RESULTSIn the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed.

CONCLUSIONSThe virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

Carcinoma, Hepatocellular ; virology ; Defective Viruses ; physiology ; Encephalitis Virus, Japanese ; chemistry ; genetics ; physiology ; Humans ; Membrane Glycoproteins ; analysis ; Mutation ; RNA Helicases ; Serine Endopeptidases ; Tumor Cells, Cultured ; Viral Envelope Proteins ; analysis ; Viral Nonstructural Proteins ; analysis

OBJECTIVETo study the effect of hepatitis C virus nonstructural protein NS(3) (HCV NS3) on telomerase activity and carcinogenesis.

METHODSStreptavidin-peroxidase (SP) conjugated method was used to detect the expression of HCV NS(3) protein in NIH3T3 cells transfected with plasmid pRcHCNS(3)-5' and pRcHCNS(3)-3'. Telomerase activity was detected by an in situ telomerase activity labeling method, telomeric repeat amplification protocol polymerase chain reaction (TRAP-PCR) and telomerase PCR enzyme linked immunosorbent assay (ELISA) technology in the transfected and non-transfected NIH3T3 cells.

RESULTSHCV NS(3) protein was expressed in the NIH3T3 cells transfected with plasmid pRcHCNS(3)-5' expressing HCV NS(3) C-terminal deleted protein or with plasmid pRcHCNS(3)-3' expressing HCV NS(3) N-terminal deleted protein. The positive signal of HCV NS(3) protein was localized in the cytoplasm of NIH3T3 cells, and the signal intensity of the former was stronger. Telomerase activity in NIH3T3 cells transfected with plasmid pRcHCNS(3)-5' was stronger than that in NIH3T3 cells transfected with plasmid pRcHCNS(3)-3' (P < 0.01), whereas telomerase activity in NIH3T3 cells transfected with plasmid pRcCMV or untreated NIH3T3 cells was weaker than that in NIH3T3 cells transfected with plasmid pRcHCNS(3)-3' (P < 0.05). The expression level of HCV NS(3) protein was significantly correlated with the strength of telomerase activity (P < 0.05). The results obtained by in situ telomerase activity labeling corresponded to the results by telomerase PCR ELISA technology.

CONCLUSIONSHCV NS(3) protein may activate telomerase through endogenous mechanism to induce host cell transformation. The effect of HCV NS(3) C-terminal deleted protein on telomerase activity in the host cell may be stronger than that of HCV NS(3) N-terminal deleted protein. In situ telomerase activity labeling was a reliable technology for studying pathological morphology and telomerase activity in tissues and cells.

3T3 Cells ; Animals ; Enzyme-Linked Immunosorbent Assay ; methods ; Mice ; Plasmids ; genetics ; Polymerase Chain Reaction ; methods ; Telomerase ; genetics ; metabolism ; Transfection ; Viral Nonstructural Proteins ; genetics ; physiology