The Study On NF-κB Activation By Hepatitis C Virus Core Protein And NS5A Protein

Hepatitis C virus (HCV) is a major causative agent for acute and chronic hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma. The mechanisms responsible for HCV-mediated chronicity and disease progression remain unclear. Nuclear factor kB (NF-κB) is a key transcription factor that activates numerals genes involved in cellular immune response and inflammation, playing an important role in promoting inflammation and in the regulation of cell proliferation and survival.In this report we have studied the regulatory interaction between HCV Core protein/NS5A protein and NF-kB to understand the mechanism responsible for HCV-mediated chronicity and disease progression.Recombinant Eukaryotic expression plasmids pcDNA-Δcore519 , pcDNA-Δcore474, pcDNA-Δcore(76-573), pcDNA-Δcore(76-474) and p3NS5A were constructed and then cotransfected with NF-κB reporter plasmid pNF-κB-Luc into Cos-7, HeLa and Huh7 three cell line. Western-Blot and Indirect immunofluorescence assay were used to detect the expression and localization of core protein mutants and NS5A protein. Luciferase assay showed that core protein can activate NF-κB in Huh7 cells but not in Cos-7 cellsor HeLa cells, suggested that the activation of NF-κB by core protein was cell type specific. Furthermore, the luciferase activity was increased in dose-dependent manner in core-expressing cells. N-terminal region (1-25aa) of core protein was important for its activation on NF-kB and the activation of NF-κB by core protein was a little related to the hydrophobility of c-terminus of core protein. When NS5A protein expressed alone, it has no obvious effect on NF-kB activation in these three cell lines. But NS5A protein can enhance full length core protein-induced NF-kB activation when co-expression with core protein. And this enhancement was related to the amount of NS5A protein.The expression levels of NF-κB/p65, NF-κB/p50 and IκB-α in C191-expressing Huh7 cells either alone or together with NS5A was studied. The results suggested that C191 alone or together with NS5A protein did not enhance the expression of NF-κB. And the NF-κB activity was augmented due to the dissociation of NF-κB-IκB complex and IκB degradation.Our results showed that the interaction of core protein and NS5A protein hassynergistic function on NF-kB activation. While the activation of NF-kB always leads to inhibition of cell apoptosis and maintain cell survival during HCV infection, thus to assist HCV replication and to maintain the low virus titre. On the other hand, activation of NF-kB will activate many cytokines and adhesion molecules related to immune and inflammatory response. So the synergistic function of core protein and NS5A protein on NF-kB activation played an important role in the establishment of chronic and persistent liver disease during HCV infection.Sever acute respiratory syndrome(SARS) coronavirus(SARS-CoV) is the major causative agent for the world wide outbreak of SARS in 2003. SARS was characterized by persistent fever, respiratory symptoms with lung consolidation, lymphopenia, and respiratory failure in life-threatening cases. The mechanisms of SARS-CoV infection caused atypical pneumonia remain unclear. The SARS-CoV nucleocapsid (N) protein is a 46-kDa structural protein. Besides its nucleocapsid assembly during the viral life cycle, N protein has also been reported to activate the activator protein 1 (API) signal transduction pathway and modulate the expression of MAPK, JNK and ERK. In this report, we studied the possible regulatory interaction of SARS-CoV N protein and NF-kB by luciferase assay.Recombinant eukaryotic expression plasmids pcNP% pcNl-225>. pcN226-422^ pcN226-300 and pcN355-422 were contransfected with cotransfected with NF-kB reporter plasmid pNF-KB-Luc into HeLa, Vero and Vero E6 three cell lines. Western-Blot was used to detect the expression of N mutants. Luciferase assay showed that SARS-CoV N protein significantly activated NF-kB in a dose-dependent manner in Vero E6 cell line that is highly susceptible to virus infection. We also found that full-length N protein had the highest activation on NF-kB. This suggested that there were more than one function domain in the N protein responsible for NF-kB activation and the activation caused by N protein is the total effect of all the function domains. While activation of NF-kB often activate the transcription of many proinflammatory molecules. This indicated that SARS-CoV N protein may be involved in the pathogenesis of SARS and this phenomenon can be considered while developing therapecutics for the SARS treatment.