title: The nuclear localization signal sequence of porcine circovirus type 2 ORF2 enhances intracellular delivery of plasmid DNA.
abstract: The phospholipid bilayer of the cell membrane is a natural barrier that prevents large molecules from entering the cell. Cationic liposomes are commonly used for transfection of plasmid DNA but they have high cost and toxicity. Many reports have shown that cell-penetrating peptides (CPP) are able to translocate across the cell membrane efficiently. The VP22 peptide of herpes simplex virus (HSV) was synthesized as a CPP. Two fusion protein candidates, containing binding/condensing protein (VP22-TmHU) and porcine circovirus type 2 nuclear localization signal (VP22-TmHU-PCV2.NLS), were constructed and expressed in E. coli in an attempt to improve delivery of plasmid DNA (pDNA). Firstly, as shown by the electrophoretic mobility shift assay (EMSA), VP22-TmHU (VT) and VP22-TmHU-PCV2.NLS (VTN) were able to bind to pDNA (pEGFP-N1) effectively. Secondly, intracellular transport of pEGFP-N1 was observed by fluorescence microscopy and quantified by flow cytometry after transfection. VTN was successful in delivering pEGFP-N1 intracellularly but VT was not. Thirdly, two protein candidates were combined with Lipofectamine, and both VT and VTN enhanced the transfection rate to 65%, compared to 25% with Lipofectamine alone. Lastly, mice were injected intramuscularly with PBS, pcDNA3-ORF2, pcDNA3-ORF2 plus Lipofectamine, pcDNA3-ORF2 plus VT, pcDNA3-ORF2 plus VT plus Lipofectamine, pcDNA3-ORF2 plus VTN, and pcDNA3-ORF2 plus VTN plus Lipofectamine. The highest level of antibodies raised against PCV2 ORF2 Cap protein was detected with pcDNA3-ORF2 plus VTN. Contrary to the in vitro results, VTN delivered pDNA effectively in vivo without Lipofectamine. In summary, the nuclear localization signal sequence of porcine circovirus type 2 ORF2 can enhance intracellular delivery of pDNA.
authors.affiliation: School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Hsu-Chung Gabriel
authors.initials: HC
authors.lastname: Chen
authors.forename: Siao-Ting
authors.initials: ST
authors.lastname: Chiou
authors.forename: Jin-Yi
authors.initials: JY
authors.lastname: Zheng
authors.forename: Shu-Hsiang
authors.initials: SH
authors.lastname: Yang
authors.forename: Shiow-Suey
authors.initials: SS
authors.lastname: Lai
authors.forename: Tsun-Yung
authors.initials: TY
authors.lastname: Kuo
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017925:Circovirus / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000008:administration & dosage / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000008:administration & dosage / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA