Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB.Methods.In vitro glutathion S-transferase (GST)resin Pull-Down assay and Far-Western Blotting assay, in vivo Co-immunoprecipition assay were used.Results.The X199(51-99)domain of HBX is reponsible for HBX binding to TFIIB.While the d10 domain (125-295)of TFIIB is required for TFIIB binding to HBX.When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L),the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost.In vitro results of HBX binding to TFIIB were further confirmed by in vivo co-immunoprecipitation assay.Our results also indicated that the Woodchuck hepatitis virus X protein (WHX)interacts with TFIIB.Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.

It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.
DNA Primers ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; physiology ; Plasmids ; Protein Binding ; RNA Replicase ; metabolism ; Recombinant Proteins ; metabolism ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Virus Replication