NDLI: The nuclear localization signal sequence of porcine circovirus type 2 ORF2 enhances intracellular delivery of plasmid DNA

Content Provider	Springer Nature Link
Author	Chen, Hsu Chung Gabriel Chiou, Siao Ting Zheng, Jin Yi Yang, Shu Hsiang Lai, Shiow Suey Kuo, Tsun Yung
Copyright Year	2011
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.
Starting Page	803
Ending Page	815
Page Count	13
File Format	PDF
ISSN	03048608
Journal	Archives of Virology
Volume Number	156
Issue Number	5
e-ISSN	14328798
Language	English
Publisher	Springer Vienna
Publisher Date	2011-02-09
Publisher Place	Vienna
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Medical Microbiology Infectious Diseases Virology
Content Type	Text
Resource Type	Article
Subject	Virology Medicine

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