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• W2885808213 abstract "Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 μm. Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 μm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions. Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 μm. Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 μm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions. Porcine circovirus (PCV), 3The abbreviations used are: PCVporcine circovirusCapcapsid proteinNLSnuclear localization signalCPPcell-penetrating peptideEGFPenhanced GFPHIV TATHIV type 1 transactivator of transcription proteinCRACcholesterol recognition amino acid consensusPIpropidium iodideGUVgiant unilamellar vesicleRFPred fluorescent proteinHSheparan sulfateHSPGheparan sulfate proteoglycanCMEclathrin-mediated endocytosisPKporcine kidney epithelialDiI1,1″-dioctadecyl-3,3,3″,3″-tetramethylindocarbocyanine perchlorateEMCCDelectron-multiplying charge-coupled device. one of the smallest animal viruses, belongs to the Circovirus genus in the family Circoviridae (1Tischer I. Gelderblom H. Vettermann W. Koch M.A. A very small porcine virus with circular single-stranded DNA.Nature. 1982; 295 (7057875): 64-6610.1038/295064a0Crossref PubMed Scopus (588) Google Scholar) and is a nonenveloped, single-stranded, and circular DNA virus. There are three genotypes of which PCV type 2 (PCV2) has been associated with various diseases, collectively termed porcine circovirus 2–associated diseases (2Segalés J. Kekarainen T. Cortey M. The natural history of porcine circovirus type 2: from an inoffensive virus to a devastating swine disease?.Vet. Microbiol. 2013; 165 (23380460): 13-2010.1016/j.vetmic.2012.12.033Crossref PubMed Scopus (140) Google Scholar), whereas PCV type 1 (PCV1) is nonpathogenic for swine (3Tischer I. Rasch R. Tochtermann G. Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines.Zentralbl. Bakteriol. Orig. A. 1974; 226 (4151202): 153-167PubMed Google Scholar). In addition, PCV type 3 (PCV3), a newly emerging circovirus, was reported in 2016, and its pathogenicity has yet to be determined (4Palinski R. Piñeyro P. Shang P. Yuan F. Guo R. Fang Y. Byers E. Hause B.M. A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure.J. Virol. 2017; 91 (27795441): e01879-e0191610.1128/JVI.01879-16Crossref PubMed Scopus (353) Google Scholar, 5Phan T.G. Giannitti F. Rossow S. Marthaler D. Knutson T.P. Li L. Deng X. Resende T. Vannucci F. Delwart E. Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation.Virol. J. 2016; 13 (27835942): 18410.1186/s12985-016-0642-zCrossref PubMed Scopus (264) Google Scholar). porcine circovirus capsid protein nuclear localization signal cell-penetrating peptide enhanced GFP HIV type 1 transactivator of transcription protein cholesterol recognition amino acid consensus propidium iodide giant unilamellar vesicle red fluorescent protein heparan sulfate heparan sulfate proteoglycan clathrin-mediated endocytosis porcine kidney epithelial 1,1″-dioctadecyl-3,3,3″,3″-tetramethylindocarbocyanine perchlorate electron-multiplying charge-coupled device. Currently, PCV2 is regarded as one of the world's most important swine pathogens, causing huge economic losses. The PCV2 genome contains two main open reading frames (ORFs). Of those, ORF1 encodes two replication-related proteins (Rep and Rep′), both of which are indispensable to virus replication, whereas ORF2 encodes capsid protein (Cap), the sole structural protein of this virus, capable of self-assembly into virus-like particles in vivo or in vitro. The NH2 terminus of the Cap, consisting of 41 residues, is the nuclear localization signal (NLS), based on subcellular localizations of various PCV2 Cap mutants, including truncated Cap (without the NLS) that was not detected in the nucleus (6Liu Q. Tikoo S.K. Babiuk L.A. Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2.Virology. 2001; 285 (11414809): 91-9910.1006/viro.2001.0922Crossref PubMed Scopus (124) Google Scholar). The NLS is enriched with positively charged residues, often present in other NLS-containing proteins. A unique signature of the NLS of the PCV Cap is an arginine-rich stretch. Interestingly, the NLS of Caps derived from other circovirus species (e.g. those affecting ducks or dogs) also contains substantial arginine residues. However, functions of these arginine residues in the NLS have not yet been confirmed. The NLS may be proximal to the icosahedral 5-fold axis of the PCV2 capsid and be involved in viral DNA packaging (7Khayat R. Brunn N. Speir J.A. Hardham J.M. Ankenbauer R.G. Schneemann A. Johnson J.E. The 2.3-angstrom structure of porcine circovirus 2.J. Virol. 2011; 85 (21632760): 7856-786210.1128/JVI.00737-11Crossref PubMed Scopus (95) Google Scholar). Therefore, the NLS might not be exposed on the capsid surface in a mature virion, although it might externalize in the metastable capsid, induced by various cellular or environmental factors during virus entry into host cells. However, roles of the NLS in PCV2 entry into host cells are unknown. The NLS of adenovirus E1A can deliver macromolecules into a nucleus by interacting with the nuclear pore complex in nondividing cells, which can further be recognized by nuclear import transporters (8Wagstaff K.M. Jans D.A. Nucleocytoplasmic transport of DNA: enhancing non-viral gene transfer.Biochem. J. 2007; 406 (17680778): 185-20210.1042/BJ20070505Crossref PubMed Scopus (68) Google Scholar, 9Douglas J.L. Quinlan M.P. Efficient nuclear localization of the Ad5 E1A 12S protein is necessary for immortalization but not cotransformation of primary epithelial cells.Cell Growth Differ. 1994; 5 (8049154): 475-483PubMed Google Scholar). Furthermore, NLS peptides derived from simian virus 40 (KKKRKV) and nucleoplasmin (KRPAAIKKAGQAKKKK) may function as cell-penetrating peptides (CPPs), delivering foreign proteins into cells, although both peptides have poor efficiencies for cellular uptake with their functional motifs rich in lysine residues (10Futaki S. Membrane-permeable arginine-rich peptides and the translocation mechanisms.Adv. Drug Deliv. Rev. 2005; 57 (15722163): 547-55810.1016/j.addr.2004.10.009Crossref PubMed Scopus (355) Google Scholar). CPP, a powerful transport vector for delivery of various cargoes through cell membranes, was first described in 1988 and is derived from a peptide of HIV type 1 transactivator of transcription protein (HIV TAT) (11Frankel A.D. Pabo C.O. Cellular uptake of the tat protein from human immunodeficiency virus.Cell. 1988; 55 (2849510): 1189-119310.1016/0092-8674(88)90263-2Abstract Full Text PDF PubMed Scopus (2320) Google Scholar). In 1997, Vivès et al. (12Vivès E. Brodin P. Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.J. Biol. Chem. 1997; 272 (9188504): 16010-1601710.1074/jbc.272.25.16010Abstract Full Text Full Text PDF PubMed Scopus (2056) Google Scholar) identified a minimal sequence with 11 residues from HIV TAT that penetrated cell membranes and entered cells. A few years later, Derossi, D. et al. (13Derossi D. Joliot A.H. Chassaing G. Prochiantz A. The third helix of the antennapedia homeodomain translocates through biological membranes.J. Biol. Chem. 1994; 269 (8144628): 10444-10450Abstract Full Text PDF PubMed Google Scholar) discovered another CPP, a 16-residue peptide of penetratin (pAntp) derived from the amphiphilic Drosophila Antennapedia homeodomain. Over the past 20 years, >100 peptides, ranging from 5 to 40 residues, have been reported to penetrate various cell membranes and carry biologically active molecules, cargoes, and compounds into cells (14Jones A.T. Sayers E.J. Cell entry of cell penetrating peptides: tales of tails wagging dogs.J. Control. Release. 2012; 161 (22516088): 582-59110.1016/j.jconrel.2012.04.003Crossref PubMed Scopus (202) Google Scholar). Among these CPPs, the HIV TAT–based delivery system has been extensively studied in cultured cells and organs (15Rapoport M. Lorberboum-Galski H. TAT-based drug delivery system—new directions in protein delivery for new hopes?.Expert Opin. Drug Deliv. 2009; 6 (19413454): 453-46310.1517/17425240902887029Crossref PubMed Scopus (82) Google Scholar). For example, by bioconjugation with imaging agents, HIV TAT can be used for intracellular imaging (16Chen B. Liu Q. Zhang Y. Xu L. Fang X. Transmembrane delivery of the cell-penetrating peptide conjugated semiconductor quantum dots.Langmuir. 2008; 24 (18823093): 11866-1187110.1021/la802048sCrossref PubMed Scopus (92) Google Scholar, 17Ruan G. Agrawal A. Marcus A.I. Nie S. Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding.J. Am. Chem. Soc. 2007; 129 (17983227): 14759-1476610.1021/ja074936kCrossref PubMed Scopus (439) Google Scholar), whereas by covalent attachment of oligonucleotides, HIV TAT can function as a vehicle to deliver foreign DNA into cells (18Abes R. Arzumanov A.A. Moulton H.M. Abes S. Ivanova G.D. Iversen P.L. Gait M.J. Lebleu B. Cell-penetrating-peptide-based delivery of oligonucleotides: an overview.Biochem. Soc. Trans. 2007; 35 (17635146): 775-77910.1042/BST0350775Crossref PubMed Scopus (102) Google Scholar). Although CPPs are efficient intracellular delivery systems, they have limitations in practical applications. First, HIV TAT was reported to move out of cells, thereby reducing intracellular concentrations of HIV TAT–attached compounds (17Ruan G. Agrawal A. Marcus A.I. Nie S. Imaging and tracking of tat peptide-conjugated quantum dots in living cells: new insights into nanoparticle uptake, intracellular transport, and vesicle shedding.J. Am. Chem. Soc. 2007; 129 (17983227): 14759-1476610.1021/ja074936kCrossref PubMed Scopus (439) Google Scholar). Second, HIV TAT and some CPPs might be trapped in endosomes and degraded during endosome maturation (19Cleal K. He L. Watson P.D. Jones A.T. Endocytosis, intracellular traffic and fate of cell penetrating peptide based conjugates and nanoparticles.Curr. Pharm. Des. 2013; 19 (23140451): 2878-289410.2174/13816128113199990297Crossref PubMed Scopus (76) Google Scholar, 20Reissmann S. Cell penetration: scope and limitations by the application of cell-penetrating peptides.J. Pept. Sci. 2014; 20 (25112216): 760-78410.1002/psc.2672Crossref PubMed Scopus (186) Google Scholar). Third, toxic side effects usually occur with HIV TAT–mediated delivery (12Vivès E. Brodin P. Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.J. Biol. Chem. 1997; 272 (9188504): 16010-1601710.1074/jbc.272.25.16010Abstract Full Text Full Text PDF PubMed Scopus (2056) Google Scholar, 15Rapoport M. Lorberboum-Galski H. TAT-based drug delivery system—new directions in protein delivery for new hopes?.Expert Opin. Drug Deliv. 2009; 6 (19413454): 453-46310.1517/17425240902887029Crossref PubMed Scopus (82) Google Scholar, 21Jones S.W. Christison R. Bundell K. Voyce C.J. Brockbank S.M. Newham P. Lindsay M.A. Characterisation of cell-penetrating peptide-mediated peptide delivery.Br. J. Pharmacol. 2005; 145 (15937518): 1093-110210.1038/sj.bjp.0706279Crossref PubMed Scopus (323) Google Scholar). Elucidating mechanisms of CPP cellular uptake should facilitate development of novel delivery vectors. It was reported that CPPs enter cells by either directly penetrating cell membranes for internalization or endocytosis-mediated translocation (22Duchardt F. Fotin-Mleczek M. Schwarz H. Fischer R. Brock R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides.Traffic. 2007; 8 (17587406): 848-86610.1111/j.1600-0854.2007.00572.xCrossref PubMed Scopus (652) Google Scholar, 23Koren E. Torchilin V.P. Cell-penetrating peptides: breaking through to the other side.Trends Mol. Med. 2012; 18 (22682515): 385-39310.1016/j.molmed.2012.04.012Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar24Brock R. The uptake of arginine-rich cell-penetrating peptides: putting the puzzle together.Bioconjug. Chem. 2014; 25 (24679171): 863-86810.1021/bc500017tCrossref PubMed Scopus (173) Google Scholar). In addition, energy-dependent macropinocytosis is considered a primary endocytosis pathway responsible for CPP-mediated intracellular delivery of various proteins (25Nakase I. Hirose H. Tanaka G. Tadokoro A. Kobayashi S. Takeuchi T. Futaki S. Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis.Mol. Ther. 2009; 17 (19707187): 1868-187610.1038/mt.2009.192Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar26Kaplan I.M. Wadia J.S. Dowdy S.F. Cationic TAT peptide transduction domain enters cells by macropinocytosis.J. Control. Release. 2005; 102 (15653149): 247-25310.1016/j.jconrel.2004.10.018Crossref PubMed Scopus (576) Google Scholar, 27Erazo-Oliveras A. Najjar K. Dayani L. Wang T.Y. Johnson G.A. Pellois J.P. Protein delivery into live cells by incubation with an endosomolytic agent.Nat. Methods. 2014; 11 (24930129): 861-86710.1038/nmeth.2998Crossref PubMed Google Scholar28Wadia J.S. Stan R.V. Dowdy S.F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.Nat. Med. 2004; 10 (14770178): 310-31510.1038/nm996Crossref PubMed Scopus (1412) Google Scholar). In that regard, dimeric HIV TAT had a crucial role in the subsequent endosomal escape of these foreign proteins into the cytoplasm (27Erazo-Oliveras A. Najjar K. Dayani L. Wang T.Y. Johnson G.A. Pellois J.P. Protein delivery into live cells by incubation with an endosomolytic agent.Nat. Methods. 2014; 11 (24930129): 861-86710.1038/nmeth.2998Crossref PubMed Google Scholar). Regardless, precise mechanisms regarding cellular uptake of various CPPs remain to be determined. In this study, NLS derived from the PCV2 Cap carried foreign protein into various cell lines. Furthermore, roles of two positively charged segments (NLS-A and -B) among the NLS were investigated: NLS-A, but not -B, may function as a novel CPP capable of entering cells. In addition, mechanisms of NLS-A cellular uptake at different concentrations were also studied by live-imaging microscopy because fixation reagents might lead to artificial redistribution due to the positively charged nature of the CPP (29Melikov K. Chernomordik L.V. Arginine-rich cell penetrating peptides: from endosomal uptake to nuclear delivery.Cell. Mol. Life Sci. 2005; 62 (16231085): 2739-274910.1007/s00018-005-5293-yCrossref PubMed Scopus (134) Google Scholar). Finally, we raised a possible model in which the NLS of the PCV Cap may have essential roles in interactions of virus–host cells and release of a viral genome into a replication site via increased membrane permeability. The NLS was strictly conserved and enriched with arginine residues within Caps of both genotypes of PCV1 and PCV2 except for 2 extra residues (33AF34) in the NLS of the PCV1 Caps (Fig. 1A). Based on secondary structure analysis, the NLS of the PCV2 Cap was composed of two stretches (NLS-A and -B) separated by an α-helix (30Wang N. Zhan Y. Wang A. Zhang L. Khayat R. Yang Y. In silico Analysis of surface structure variation of PCV2 capsid resulted from loops mutation of its capsid protein (Cap).J. Gen. Virol. 2016; 97 (27902320): 3331-334410.1099/jgv.0.000634Crossref PubMed Scopus (16) Google Scholar), although this helix was absent in the PCV3 Cap (Fig. 1A). In PCV2, NLS-A contained 9 arginine residues, whereas NLS-B had 8 arginines and 1 lysine residue (Fig. 1, A and C). Furthermore, NLS derived from various circovirus species all had a signature motif rich in arginine residues (Fig. 1B). In addition, there was a well known and conserved motif of the cholesterol recognition amino acid consensus (CRAC) in PCV2 Caps (Fig. 1C). The CRAC motif, comprising -(L/V)X1–5YX1–5(R/K)- where X1–5 represents 1–5 residues of any amino acid at the position(s) (31Levitan I. Singh D.K. Rosenhouse-Dantsker A. Cholesterol binding to ion channels.Front. Physiol. 2014; 5 (24616704): 6510.3389/fphys.2014.00065Crossref PubMed Scopus (140) Google Scholar), was only in NLS-B of PCV2 Caps, but not in PCV1, due to insertion of 2 residues in NLS-B (Fig. 1A). To determine whether the arginine-rich NLS of the PCV2 Cap can carry foreign protein across cell membranes and enter cells in vitro, porcine kidney epithelial (PK15) (Fig. 2, A–C) or Sf9 cells (Fig. 2, D–F) were incubated with purified NLS-fused EGFP (NLS–EGFP) or EGFP (Fig. S1), respectively. The NLS–EGFP entered both cell lines (Fig. 2, A and D, left panels). In contrast, the fluorescence in EGFP-treated cells was significantly low (Fig. 2, A and D, middle panels). Flow cytometry analysis demonstrated that both PK15 cells and Sf9 cells incubated with NLS–EGFP had a significantly increased number of cells with intracellular fluorescence (Fig. 2, B and E) and higher mean fluorescence intensity (Fig. 2, C and F) compared with PK15 cells or Sf9 cells incubated with EGFP or the controls. Both NLS-A and -B are rich in positively charged residues and form two independent stretches divided by an α-helix (30Wang N. Zhan Y. Wang A. Zhang L. Khayat R. Yang Y. In silico Analysis of surface structure variation of PCV2 capsid resulted from loops mutation of its capsid protein (Cap).J. Gen. Virol. 2016; 97 (27902320): 3331-334410.1099/jgv.0.000634Crossref PubMed Scopus (16) Google Scholar) (Fig. 1A). Numbers of positively charged residues within each were comparable with two known CPPs (HIV TAT and polyarginine Arg8) (Fig. 1C). We hypothesized that both NLS-A and -B may function as CPPs. To test this hypothesis, both peptides were synthesized, conjugated with FITC, and incubated with PK15 cells. NLS-A (Fig. 3A, upper row) entered PK15 cells, whereas NLS-B failed to enter cells (Fig. 3A, middle row). Flow cytometry analysis demonstrated that PK15 cells incubated with NLS-A had a significantly higher percentage of cells with intracellular fluorescence (Fig. 3B) and higher mean fluorescence intensity (Fig. 3C) than PK15 cells incubated with NLS-B or the control. In addition, besides PK15, both NLS-A and HIV TAT also entered HeLa and NIH3T3 cells (Fig. 4A). Flow cytometry analysis (Fig. 4B) was consistent with the above confocal imaging result.Figure 4.Cellular uptake of FITC-conjugated NLS-A and FITC-conjugated HIV-TAT. A, PK15, HeLa, and NIH3T3 cells were incubated with FITC-conjugated NLS-A (upper row) or FITC-conjugated HIV-TAT (middle row) for 30 min and washed three times with PBS before imaging with a dual-channel confocal laser-scanning microscope (Nikon TiE). A third test with buffer only was used as a treatment control (bottom row). Green indicates FITC signal in the cells, and cell nuclei (blue) are indicated by Hoechst. B, bar graph summarizing the fluorescence intensity of the PK15, HeLa, and NIH3T3 cells under the above three incubation treatments (n = 4; error bars represent S.D.; ***, p < 0.001). All scale bars represent 10 μm. a.u., arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) There was a punctate distribution pattern of NLS-A in cells after peptide internalization irrespective of peptide concentration in culture (Fig. 5A). Therefore, NLS-A may enter cells via endocytosis and localize within vesicles. However, an even distribution of the NLS-A throughout the cell cytoplasm, along with the punctate pattern, was also observed when peptide concentration in culture was ≥10 μm (Fig. 5A), implying a possible alternative concentration-dependent entry mechanism of NLS-A. In addition, flow cytometry analysis demonstrated that cellular uptake of NLS-A increased with elevated concentrations of this peptide in cell culture (Fig. 5, B and C). To exclude extracellular fluorescence (e.g. membrane surface–associated NLS-A), PK15 cells incubated with peptides or proteins were always treated with trypsin at 37 °C for 2 min (see “Materials and methods”) before flow cytometry analysis (because NLS-A was sensitive to trypsinization; Fig. S2). In previous reports, CPPs had variable cytotoxicity at high concentration (12Vivès E. Brodin P. Lebleu B. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.J. Biol. Chem. 1997; 272 (9188504): 16010-1601710.1074/jbc.272.25.16010Abstract Full Text Full Text PDF PubMed Scopus (2056) Google Scholar, 15Rapoport M. Lorberboum-Galski H. TAT-based drug delivery system—new directions in protein delivery for new hopes?.Expert Opin. Drug Deliv. 2009; 6 (19413454): 453-46310.1517/17425240902887029Crossref PubMed Scopus (82) Google Scholar, 21Jones S.W. Christison R. Bundell K. Voyce C.J. Brockbank S.M. Newham P. Lindsay M.A. Characterisation of cell-penetrating peptide-mediated peptide delivery.Br. J. Pharmacol. 2005; 145 (15937518): 1093-110210.1038/sj.bjp.0706279Crossref PubMed Scopus (323) Google Scholar); accordingly, we also tested the cytotoxicity of NLS-A. Cells were treated with high concentration of NLS-A (40 μm) for 30 min followed by propidium iodide (PI) staining. As shown in Fig. 5D and Fig. S3, >90% of the cells were viable with incubation of NLS-A, even at a high concentration of 40 μm. Furthermore, we compared the cytotoxicity of NLS-A and HIV TAT upon PK15 cells at various concentrations. More than 95% of PK15 cells were viable under incubation of both CPPs that were lower than 20 μm (Fig. S3). As suggested above in Fig. 5, the entry mechanism of NLS-A might be concentration-dependent. To further dissect the scenario, we monitored the NLS-A entry process by time-lapse confocal microscopy at high concentrations (40 μm). The increasing intracellular fluorescence signal was recorded every 5 s after the addition of peptide to cell culture (Fig. 6A), and the quantified intracellular mean fluorescence intensity was plotted against time (Fig. 6B). The rapid cellular uptake and even distribution of NLS-A suggested that the peptide may enter cells either through a receptor-independent pathway or by directly penetrating the cell membranes at high concentrations. To test whether NLS-A induced membrane permeability, we mixed NLS-A with giant unilamellar vesicles (GUVs) with encapsulated fluorescent dye (atto565) and monitored the GUVs with confocal microscopy. Fluorescence (red) within the GUVs turned green after NLS-A was mixed with the GUVs (Fig. 6, C and D), demonstrating the entry of NLS-A into the GUVs; in addition, the dye (red) in the GUVs also leaked out from the vesicles into the buffer. This result strongly indicated that NLS-A increased the permeability of the membrane and led to the equilibration of NLS-A and the atto565 dye across membranes (Fig. 6D). However, in the presence of FITC alone (Fig. 6E) or in the absence of NLS-A (Fig. 6C), the atto565 dye within the GUVs was quite stable. Statistical analysis also demonstrated that the ratio of GUVs containing green dye in the presence of NLS-A was significantly higher than that in the presence of FITC alone (Fig. 6F; p < 0.001). Therefore, a high concentration of NLS-A permeabilized the GUV membrane and caused a bidirectional flux of the dye across the membrane. It appears that HIV TAT and other CPPs enter cells via endocytosis with distinct pathways (22Duchardt F. Fotin-Mleczek M. Schwarz H. Fischer R. Brock R. A comprehensive model for the cellular uptake of cationic cell-penetrating peptides.Traffic. 2007; 8 (17587406): 848-86610.1111/j.1600-0854.2007.00572.xCrossref PubMed Scopus (652) Google Scholar). The punctate distribution of the NLS-A at low concentrations indicated that the peptide was translocated into cells via endocytosis. Dynamic tracking of NLS-A in live cells indicated that the NLS-A colocalized with Rab7–RFP in PK15 cells (Fig. 7A). Therefore, NLS-A was present in a late endosome after internalization. Next, we applied endocytosis inhibitors to cell cultures to further study the NLS-A entry mechanism. Inhibition of clathrin-mediated endocytosis (chlorpromazine) and macropinocytosis (N-(ethyl-N-isopropyl)-amiloride) and disruption of caveolae/lipid rafts (methyl-β-cyclodextrin) all dramatically decreased efficiency of NLS-A entry into cells (Fig. 7, B and C). Therefore, the three major endocytosis pathways were all involved in internalization of NLS-A at low concentration (6 μm). In addition, effects of F-actin on NLS-A internalization were also tested. Inhibition of actin polymerization by cytochalasin D dramatically decreased efficiency of NLS-A (6 μm) entry into PK15 cells (Fig. S4), which implicated F-actin to have a critical role in NLS-A internalization. Finally, we compared the entry efficiency of NLS-A and HIV TAT. PK15 cells were incubated with FITC-conjugated NLS-A or HIV TAT at various concentrations, and cellular uptake efficiencies of the peptides were evaluated by flow cytometry. Mean fluorescence intensities were compared between NLS-A and HIV TAT at various concentrations, and NLS-A entry into PK15 cells was 10-fold more efficient than entry of HIV TAT in PK15 cells (Fig. 8). Similar results were also observed in HeLa cells (Fig. S5). In a mature PCV2 virion, viral genomic DNA is packaged by a capsid, assembled by 60 Cap subunits. The NLS of the PCV2 Cap is considered a critical element responsible for viral DNA packaging as the NLS, which is rich in positively charged residues, may interact with phosphate groups of viral DNA. Mutation of the NLS significantly decreases virus titers in cell culture (32Shuai J. Fu L. Zhang X. Zhu B. Li X. He Y. Fang W. Functional exchangeability of the nuclear localization signal (NLS) of capsid protein between PCV1 and PCV2 in vitro: implications for the role of NLS in viral replication.Virol. J. 2011; 8 (21733152): 34110.1186/1743-422X-8-341Crossref PubMed Scopus (12) Google Scholar, 33Beach N.M. Smith S.M. Ramamoorthy S. Meng X.J. Chimeric porcine circoviruses (PCV) containing amino acid epitope tags in the C terminus of the capsid gene are infectious and elicit both anti-epitope tag antibodies and anti-PCV type 2 neutralizing antibodies in pigs.J. Virol. 2011; 85 (21307200): 4591-459510.1128/JVI.02294-10Crossref PubMed Scopus (25) Google Scholar). In addition, based on a structural study, the NLS of the PCV2 Cap is apparently located around the 5-fold axis and buried inside the capsid (7Khayat R. Brunn N. Speir J.A. Hardham J.M. Ankenbauer R.G. Schneemann A. Johnson J.E. The 2.3-angstrom structure of porcine circovirus 2.J. Virol. 2011; 85 (21632760): 7856-786210.1128/JVI.00737-11Crossref PubMed Scopus (95) Google Scholar). Perhaps NLSs from five neighboring PCV2 Caps at one icosahedral 5-fold axis of the capsid may be transiently externalized and penetrate cell membran" @default.
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