Designed Artificial Transcription Factors Inhibit Hepatitis B Virus Transcription

ObjectiveArtificial transcription factor was designed to target HBV corepromoter and inhibit the transcriprion of viral gene.Methods1. The sequence of HBV core promoter was submitted to the ZincFinger Tools and selected a sequence from the core preomoter as the targetsite of the ZFP. The amino acid sequence of the ZFP was analyzed by theSwiss Model and ExPASy Proteomics tools.2. The nucleic acid sequence of the ZFP was synthesised and clonedinto the pUC57vector. ZFP was amplified by PCR with the primers thatcontain a nuclear localization signal at the5’ end and cloned into theeukaryotic expression vector pcDNA3.1(+) using BamH1and EcoRVrestriction endonuclease sites. Then, using the restriction enzymes EcoRVand XhoI, we added the KRAB domain in-frame downstream of the ZFP.The resulting plasimd was named pcDNA3.1(+)-ATF.3. Recombinant plasmid pcDNA3.1(+)-ATF was transfected intoCOS-7cell by LipofectamineTM2000. The expression of ATF was detected by reverse-transcript PCR (RT-PCR) and Western Blot.4. Recombinant plasmid pcDNA3.1(+)-ATF was transfected intoHepG2.2.15cell. The level of HBV DNA in cell culture mediums wasquantified by Real-time PCR. The viral proteins were detected in transfectedcells by ELSIA for HBV e antigen(HBeAg) and immunocytochemistry forHBV c antigen(HBcAg). Intracellular HBV mRNA was reverse transcribedand quantified by RT-PCR.Results1. A18bp length sequence(5’-AATGTCAACGACCGACCT-3’) wasselected from core promoter as the targer site of the zinc finger protein.2. Then amino acid saequence of zinc finger progtein was obtained:LEPGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPYKCPECGKSFSQSGHLTEHQRTHTGEKPYKCPECGKSFSDPGNLVRHQRTHTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSTTGNLTVHQRTHTGKKTS3. The main feature of this zinc finger protein: theoretical isoelectricpoint9.20, molecular weight19.69, instability index31.41. Both SequenceFeature Scan and homology modeling indicated that the zinc finer proteincontained six zinc finger motifs. Structure Assessment indicated thestructure of the zinc finger protein was theoretically stable.4. The expression of mRNA and protein increased evidently in COS-7cells transfected with pcDNA3.1(+)-ATF. 5. In ATF-expression cells, viral RNA and DNA was significantlyreduced, and viral proteins were dramatically decreased, indicating that ATFcould inhibit the transcription of HBV in vitro.ConclusionThese results show that ATF treatment targeting HBV core proteinpromoter has an antiviral effect and inhibits HBV infection in host cells;these results further suggest that designing new artificial transcriptionfactors may be a valuable antiviral therapy to treat HBV patients.