[Hepatitis C virus NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell].

OBJECTIVE: To construct the point mutation plasmids expressing NS3/4A with different secondary structure of the amino-terminal 120 residues of NS3, and to investigate the differences in serine protease and inhibitory effects on host cells between each subgroup. METHODS: The point mutation plasmids were constructed, which expressed NS3/4A with the corresponding secondary structures of subgroup, and were named as A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, and B2-2, with the backbone of M-H05-5 (A1-1). Western blot was performed to detect the expression of NS3/4A and the difference in in cis and in trans NS3 serine protease activity between each subgroup. The inhibitory effects of HCV NS3/4A with different amino-terminal secondary structures on IFN-beta production and p53-dependent transcriptional activation were revealed by Luciferase reporter assay. RESULTS: Western blot revealed the successful expression of the constructs and the incomplete cleavage of NS3/4A in subgroup A2-1 and B2-1, indicating that the in cis NS3 serine protease activities of subgroup A2-1 and B2-1 were weaker compared with that of the other subgroups. By using NS5A/5BDeltaC as a substrate for NS3/4A serine protease, it was also found that the in trans NS3 serine protease activities of subgroup A2-1 and B2-1 were also weaker compared with that of the other subgroups. Differences in inhibitory effects of HCV NS3 on IFN-beta promoter activity and on p53-dependent luciferase gene transcriptional activation were also observed between subgroup A2-1, B2-1 and the other subgroups. CONCLUSION: HCV NS3/4A with different secondary structures at amino-terminus has different serine protease activities and inhibitory activities on host cell functions.

[Construction of pSG5/NS5A5BdeltaC and its expression in Huh7 cells].

AIM: To construct the plasmid pSG5/NS5A5BdeltaC, and to investigate its expression in Huh7 cells. METHODS: The plasmidpTM1-NS2NS5A5BdeltaC was taken as the template. The primers were designed according to the characteristics of the sequence and endoenzyme cleavage sites in HCV NS5A5BdeltaC and vector pTM1, pSG5. The HCV NS5A5BdeltaC gene fragment was amplified by PCR from pTM1-NS2NS5A5BdeltaC and inserted into vector pSG5. The positive clones were screened by Ampicillin and identified by the restriction endoenzyme Sac I and Bgl II digestion and agarose gel electrophoresis. The constructs were transfected into Huh7 cells with FuGene 6 reagents. Immunofluorescence and Western blot were performed to detect the expression of the constructs in Huh7 cells. RESULTS: Endoenzyme digestion analysis showed that the size and the inserting orientation of the fragment met the design expectation. Immunofluorescence staining displayed the expression of HCV NS5A5BdeltaC protein, which was located in the cytoplasm, and the expression rate reached as high as 40%. SDS-PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/NS5A5BdeltaC was about 82 ku, which was consistent with the theoretical value. CONCLUSION: pSG5/NS5A5BdeltaC is successfully constructed, and it can be expressed transiently in Huh7 cells, which would lay a foundation for the further study on function of HCV polyprotein.

Construction of point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal and their expressions in Huh 7 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.</p><p><b>METHODS</b>Using pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs.</p><p><b>RESULTS</b>Indirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes.</p><p><b>CONCLUSION</b>The point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.</p>

Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway.

We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk.

Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-gamma1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities.