Effect Of HIV-1 Tat On HCV RNA Replication And Protein Expression

Objective:Both human immunodeficiency virus(HIV)and hepatitis C virus(HCV)infections are distributed worldwide.Because of the same transmission routes,there is a high rate of HIV/HCV co-infection,and they exert the mutual impact on their clinical outcomes, but the mechanism remains unclear.HIV Tat protein is an important regulatory protein,which can not only trans-activate the transcription of HIV,but also stimulates the replication and transcription of other virus.This study was designed to construct a eukaryotic expression plasmid of HIV-1 Tat protein and then to explore the influence of HIV-1 Tat protein on HCV RNA replication and protein expression.Methods:DNA fragment encoding HIV-1 Tat protein was amplified by PCR from the plasmid pNL4-3.Then the PCR product was digested and inserted into pcDNA3.1(+),an eukaryotic expression plasmid,to generate a recombinant plasmid,designated as pcDNA3.1-tat.The resulting plasmid was verified by restriction enzyme mapping and direct DNA sequencing,and then was transfected into huh-7 cells by lipo-fectamine.RT-PCR and Western blot were used to identify whether the object protein was correctly expressed in huh-7 cells.The full-length HCV replicon pcDNA6/TR-Tight/JFH1/AR and pcDNA3.1-tat were co-transfected into eukaryotic huh-7 cells by lipo-fectamine.The replication and expression of HCV RNA were detected by RT-PCR and Western blot respectively.Results:The results showed that the 240 bp DNA fragment of tat was correctly amplified and the recombinant plasmid pcDNA3.1-tat was successfully constructed,which could be correctly expressed in the huh-7 cells.And the results of RT-PCR showed that there were significant differences between test groups and control groups(P＜0.05),meaning HIV-1 Tat protein enhanced HCV RNA replication.The results of Western blot indicated that HIV-1 Tat protein up-regulated HCV core protein expression.Conclusions:Our data suggest that HIV-1 Tat can enhance the replication and protein expression of HCV RNA.This study lays the experimental and theoretical foundation for further study on the molecular mechanisms of HIV/HCV interaction which enhances HCV pathogenicity,and provides a new experimental basis for the prevention and treatment of HCV infection of HIV carriers.