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Protein & Peptide Letters


ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

Research Article

Anti-viral Effects of Superpositively Charged Mutant of Green Fluorescent Protein

Author(s): Rouhollah Vahabpour, Parya Basimi, Farzin Roohvand, Hassan Asadi, Gholnaz M. Irani, Rezvan Zabihollahi and Azam Bolhassani*

Volume 26 , Issue 12 , 2019

Page: [930 - 939] Pages: 10

DOI: 10.2174/0929866526666190823145916

Price: $65


Background: Supercharged GFP proteins were known as effective carriers for delivery of macromolecules into eukaryotic cells as well as fluorescent fusion tags for in vitro and in vivo detection.

Objective: Herein, anti-viral effects of +36 GFP and its anti-tumor effects were studied in vitro and in vivo, respectively.

Methods: We evaluated anti-HIV, anti-HSV, and anti-HCV effects of +36 GFP in vitro using ELISA, and real time PCR as common techniques for their detection, respectively. Moreover, we assessed the role of +36 GFP for eliciting HPV-related anti-tumor effects in mice due to the lack of HPV replication in vitro.

Results: Our data showed that +36 GFP efficiently enter the cells and augment the transfection rate of HPV16E7 antigen, as well. Furthermore, +36 GFP significantly reduced HCV, HIV and HSV replication up to 75%, 49% and 43% in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells, respectively. On the other hand, mice immunization with +36 GFP complexed with HPV16 E7 antigen (+36GFP + E7) or fused to HPV16 E7 antigen (+36GFP-E7) elicited a higher Th1 cellular immune response with the predominant IgG2a, IgG2b, IFN-γ and Granzyme B levels than those induced by other groups. These regimens protected mice against TC- 1 tumor challenge (~ 67%) compared to E7 protein alone (~ 33%). These data suggested that +36 GFP can act as an anti-viral agent at certain dose due to its high efficiency in cell penetration in vitro and in vivo.

Conclusion: Generally, +36 GFP targets viral replication in vitro as well as helps to suppress the growth of HPV-related tumors in vivo.

Keywords: Supercharge protein, +36 GFP, sexual viruses, anti-viral effect, anti-tumor property, viral replication.

Graphical Abstract
Ganim, Z.; Rief, M. Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding. Proc. Natl. Acad. Sci. USA, 2017, 114(42), 11052-11056.
[] [PMID: 29073015]
Nogales, A.; Baker, S.F.; Martínez-Sobrido, L. Replication-competent influenza A viruses expressing a red fluorescent protein. Virology, 2015, 476, 206-216.
[] [PMID: 25553516]
Dal Pozzo, F.; Andrei, G.; Daelemans, D. Winkler, M.; Piette, J.; De Clercq, E.; Snoeck, R. . Fluorescence-based antiviral assay for the evaluation of compounds against vaccinia virus, varicella zoster virus and human cytomegalovirus. J. Virol. Methods, 2008, 151(1), 66-73.
[] [PMID: 18490063]
Xu, M.; Jiao, Y.Y.; Fu, Y.H.; Jiang, N.; Zheng, Y.B.; Yan, Y.F.; Zhang, M.; Zheng, Y.P.; Zhu, W.Y.; Peng, X.L.; He, J.S. Construction and characterization of a recombinant human respiratory syncytial virus encoding enhanced green fluorescence protein for antiviral drug screening assay. BioMed Res. Int., 2018, 20188431243
[] [PMID: 29568767]
Leardkamolkarn, V.; Sirigulpanit, W. Establishment of a stable cell line coexpressing Dengue virus-2 and green fluorescent protein for screening of primary screening of anti-DENV compounds. J. Biomol. Screen., 2012, 17(3), 283-292.
[] [PMID: 22068705]
Mukherjee, A.; Schroeder, C.M. Flavin-based fluorescent proteins: emerging paradigms in biological imaging. Curr. Opin. Biotechnol., 2015, 31, 16-23.
[] [PMID: 25151058]
McNaughton, B.R.; Cronican, J.J.; Thompson, D.B.; Liu, D.R. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc. Natl. Acad. Sci. USA, 2009, 106(15), 6111-6116.
[] [PMID: 19307578]
Thompson, D.B.; Cronican, J.J.; Liu, D.R. Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells. Methods Enzymol., 2012, 503, 293-319.
[] [PMID: 22230574]
Bar-Shir, A.; Liang, Y.; Chan, K.W.Y.; Gilad, A.A.; Bulte, J.W.M. Supercharged green fluorescent proteins as bimodal reporter genes for CEST MRI and optical imaging. Chem. Commun. (Camb.), 2015, 51(23), 4869-4871.
[] [PMID: 25697683]
Cronican, J.J.; Thompson, D.B.; Beier, K.T.; McNaughton, B.R.; Cepko, C.L.; Liu, D.R. Potent delivery of functional proteins into Mammalian cells in vitro and in vivo using a supercharged protein. ACS Chem. Biol., 2010, 5(8), 747-752.
[] [PMID: 20545362]
Thrift, A.P.; El-Serag, H.B.; Kanwal, F. Global epidemiology and burden of HCV infection and HCV-related disease. Nat. Rev. Gastroenterol. Hepatol., 2017, 14(2), 122-132.
[] [PMID: 27924080]
Imran, M.; Waheed, Y.; Ghazal, A.; Ullah, S.; Safi, S.Z.; Jamal, M.; Ali, M.; Atif, M.; Imran, M.; Ullah, F. Modern biotechnology-based therapeutic approaches against HIV infection. Biomed. Rep., 2017, 7(6), 504-507.
[] [PMID: 29250325]
Yu, X.; He, S. The interplay between human herpes simplex virus infection and the apoptosis and necroptosis cell death pathways. Virol. J., 2016, 13, 77.
[] [PMID: 27154074]
Yang, A.; Jeang, J.; Cheng, K.; Cheng, T.; Yang, B.; Wu, T.C.; Hung, C.F. Current state in the development of candidate therapeutic HPV vaccines. Expert Rev. Vaccines, 2016, 15(8), 989-1007.
[] [PMID: 26901118]
Yang, A.; Farmer, E.; Wu, T.C.; Hung, C.F. Perspectives for therapeutic HPV vaccine development. J. Biomed. Sci., 2016, 23(1), 75.
[] [PMID: 27809842]
Kristensen, M.; Birch, D.; Mørck Nielsen, H. Applications and challenges for use of cell-penetrating peptides as delivery vectors for peptide and protein cargos. Int. J. Mol. Sci., 2016, 17(2), 1-17.
[] [PMID: 26840305]
Saade, F.; Petrovsky, N. Technologies for enhanced efficacy of DNA vaccines. Expert Rev. Vaccines, 2012, 11(2), 189-209.
[] [PMID: 22309668]
Motevalli, F.; Khodaei, A.; Anvari, M.; Bolhassani, A. Generation of a fluorescent oncoprotein in soluble form and its delivery into mammalian cells. Bratisl. Lek Listy, 2019, 120(2), 106-112.
[] [PMID: 30793613]
Motevalli, F.; Bolhassani, A.; Hesami, S.; Shahbazi, S. Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo. Immunol. Lett., 2018, 194, 29-39.
[] [PMID: 29273425]
Radcliffe, P.A.; Mitrophanous, K.A. Multiple gene products from a single vector: ‘self-cleaving’ 2A peptides. Gene Ther., 2004, 11, 1673-1674.
Szymczak, A.L.; Workman, C.J.; Wang, Y.; Vignali, K.M.; Dilioglou, S.; Vanin, E.F.; Vignali, D.A. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat. Biotechnol., 2004, 22(5), 589-594.
[] [PMID: 15064769]
Liu, Z.; Chen, O.; Wall, J.B.J.; Zheng, M.; Zhou, Y.; Wang, L.; Ruth Vaseghi, H.; Qian, L.; Liu, J. Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep., 2017, 7(1), 2193.
[] [PMID: 28526819]
Bolhassani, A.; Zahedifard, F.; Taghikhani, M.; Rafati, S. Enhanced immunogenicity of HPV16E7 accompanied by Gp96 as an adjuvant in two vaccination strategies. Vaccine, 2008, 26(26), 3362-3370.
[] [PMID: 18471945]
Kadkhodayan, S.; Sadat, S.M.; Irani, S.; Fotouhi, F.; Bolhassani, A. Generation of GFP native protein for detection of its intracellular uptake by cell-penetrating peptides. Folia Biol. (Praha), 2016, 62(3), 103-109.
[PMID: 27516189]
Soleymani, S.; Zabihollahi, R.; Shahbazi, S.; Bolhassani, A. Antiviral effects of saffron and its major ingredients. Curr. Drug Deliv., 2018, 15(5), 698-704.
[] [PMID: 29189153]
Wakita, T.; Pietschmann, T.; Kato, T.; Date, T.; Miyamoto, M.; Zhao, Z.; Murthy, K.; Habermann, A.; Kräusslich, H.G.; Mizokami, M.; Bartenschlager, R.; Liang, T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med., 2005, 11(7), 791-796.
[] [PMID: 15951748]
Zabihollahi, R.; Sadat, S.M.; Vahabpour, R.; Aghasadeghi, M.R.; Memarnejadian, A.; Ghazanfari, T.; Salehi, M.; Rezaei, A.; Azadmanesh, K. Development of single-cycle replicable human immunodeficiency virus 1 mutants. Acta Virol., 2011, 55(1), 15-22.
[] [PMID: 21434701]
Li, Y.; Subjeck, J.; Yang, G.; Repasky, E.; Wang, X.Y. Generation of anti-tumor immunity using mammalian heat shock protein 70 DNA vaccines for cancer immunotherapy. Vaccine, 2006, 24(25), 5360-5370.
[] [PMID: 16714072]
Chapman, A.M.; McNaughton, B.R. Scratching the surface: Resurfacing proteins to endow new properties and function. Cell Chem. Biol., 2016, 23(5), 543-553.
[] [PMID: 27203375]
Wu, Q.; Cheng, Q.; Yuan, S.; Qian, J.; Zhong, K.; Qian, Y.; Liu, Y. A cell-penetrating protein designed for bimodal fluorescence and magnetic resonance imaging. Chem. Sci. (Camb.), 2015, 6(11), 6607-6613.
[] [PMID: 28757964]
Crone, D.E.; Huang, Y.M.; Pitman, D.J.; Schenkelberg, C.; Fraser, K.; Macari, S.; Bystroff, C. GFP-based biosensors; Intech Open, 2013.
Shahbazi, S.; Bolhassani, A. Comparison of six cell penetrating peptides with different properties for in vitro and in vivo delivery of HPV16 E7 antigen in therapeutic vaccines. Int. Immunopharmacol., 2018, 62, 170-180.
[] [PMID: 30015237]
Margie, L. The discovery and characterization of endosomal escape enhancing compounds to improve protein delivery efficacy. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences,. 2015., 1-115.

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