The Analysis Of The Replication Activity Of NS5B Proteins Using 3' Terminuses Of Negative RNA Strand From Defferent HCV Genotypes As Templates

OBJECTIVE : to analyse the replication activity of NS5B proteins of HCV genotype 1a and 1b when use 3'terminuses of negative RNA strand from HCV 1a, 1b and 2a as templates by performing RdRp assay in vitro and offer some useful data to optimize the model for the screening of anti-HCV drugs targeting at NS5B.METHODS: The recombinant plasmid pETHis5B2 prokaryon-expressing HCV (1b) NS5B protein that the hydrophobic C-terminal 21 animo acids were deleted was constructed and transformed into Escherichia coli BL21(DE3) with the recombinant plasmid pETHis5B1 harboring cDNA sequence of HCV (1a) NS5B protein(also deleting C-terminal 21 animo acids). The soluble NS5B proteins (both genotype 1a and 1b) were expressed and purified in the same conditions. The purified NS5B proteins were confirmed by Western blot analysis and their RdRp activity was detected through dot-blot and RT-PCR. The DNA sequences corresponding to the 3'terminuses of HCV genotype 1a, 1b and 2a negative RNA strand were cloned to the transcription plasmid pGEM3Zf(+). The 3'terminuses of negative RNA strand from 1a, 1b and 2a were prepared via in vitro transcription, using these RNA as temlates and the two purified NS5B proteins as polymerase respectively we conducted the RdRp assay in vitro. Northern blot was employed to detect the RdRp products.RESULTS: Prokaryon-expressed and soluble NS5B proteins were expressed and purified effectively. The purified proteins had RdRp activity, and could synthesize RNA products similar in length to the RNA templates using the 3'terminuses of HCV genotype 1a, 1b and 2a negative RNA strand. The two NS5B proteins showed the same RdRp activity changes to these three templates: they had the highest activity using 3'terminuses from HCV genotype 2a negative RNA strand as template and the lowest activity using 3'terminuses of HCV genotype 1b negative RNA strand as template.CONCLUSION: The prokaryon-expressed NS5B proteins that the C-terminal 21 amino acids were deleted have good solubility, excellent purified effect, and the RdRp activity in vitro. The initiation of RNA synthesis belongs to do novo fashion when NS5B protein uses the negative RNA strand as template.The NS5B protein's RdRp activity is distinct when use different genetic-coming 3'terminuses of negative RNA strand as templates.