Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer

Gene Therapy volume 13, pages 821–836 (2006)Cite this article

Abstract

The adenovirus (Ad) penton base protein facilitates viral infection by binding cell surface integrins, triggering receptor-mediated endocytosis and mediating endosomal penetration. Given these multiple functions, recombinant penton base proteins have been utilized as non-viral vehicles for gene transfer by our lab and others. Although we have previously demonstrated that penton base-derived vectors undergo integrin-specific binding and cell entry, less than desirable levels of gene expression have led us to re-evaluate the recombinant penton base as an agent for gene delivery. To do so, we have examined here the intracellular trafficking of an Ad serotype 5 (Ad5) recombinant penton base protein (PB). Here, we not only observed that PB utilizes a similar, typical trafficking pathway of whole Ad, but also found that PB entered HeLa cells through pathways not yet identified as contributing to cell entry by the whole virus. We show by high-resolution confocal microscopy and biochemical methods that binding to αv-integrins is a requirement for cell entry, but that early internalization stages did not substantially pass through clathrin-positive and early endosomal compartments. Moreover, a subpopulation of internalized protein localized with caveolin-positive compartments and Golgi markers, suggesting that a certain percentage of proteins pass through non-clathrin-mediated pathways. Similar to the virus, trafficking toward the nucleus was affected by disruption of microtubules and dynein. The majority of penton base molecules avoided the lysosome while facilitating early vesicle release of low molecular weight dextran molecules. In further support of a vesicle escape capacity, a subpopulation of internalized penton base appeared to enter the nucleus, as observed by high-resolution confocal microscopy and cell fractionation. As a confirmation of these findings, we demonstrate that a recombinant penton base facilitated cytosolic entry of an siRNA molecule as observed by RNA interference of a marker gene. Based on our findings here, we suggest that whereas soluble penton base proteins may enter cells through clathrin- and non-clathrin-mediated pathways, vesicle escape and nuclear delivery appear to be supported by a clathrin-mediated pathway. As our previous efforts have focused on utilizing recombinant penton base proteins as delivery agents for therapeutics, these findings allow us to evaluate the use of the penton base as a cell entry and intracellular trafficking agent, and may be of interest concerning the development of vectors for efficient delivery of therapeutics to cells.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9

Similar content being viewed by others

References

  1. Medina-Kauwe LK . Endocytosis of adenovirus and adenovirus capsid proteins. Adv Drug Deliv Rev 2003; 55: 1485–1496.

    Article  CAS  PubMed  Google Scholar 

  2. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR . Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment. Cell 1993; 73: 309–319.

    Article  CAS  PubMed  Google Scholar 

  3. Karayan L, Hong SS, Gay B, Tournier J, d'Angeac AD, Boulanger P . Structural and functional determinants in adenovirus type 2 penton base recombinant protein. J Virol 1997; 71: 8678–8689.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997; 275: 1320–1323.

    Article  CAS  PubMed  Google Scholar 

  5. Novelli A, Boulanger PA . Deletion analysis of functional domains in baculovirus-expressed adenovirus type 2 fiber. Virology 1991; 185: 365–376.

    Article  CAS  PubMed  Google Scholar 

  6. Devaux C, Adrian M, Berthet-Colominas C, Cusack S, Jacrot B . Structure of adenovirus fibre. I. Analysis of crystals of fibre from adenovirus serotypes 2 and 5 by electron microscopy and X-ray crystallography. J Mol Biol 1990; 215: 567–588.

    Article  CAS  PubMed  Google Scholar 

  7. Boudin ML, Boulanger P . Assembly of adenovirus penton base and fiber. Virology 1982; 116: 589–604.

    Article  CAS  PubMed  Google Scholar 

  8. Rentsendorj A, Agadjanian H, Chen X, Cirivello M, Macveigh M, Kedes L et al. The Ad5 fiber mediates nonviral gene transfer in the absence of the whole virus, utilizing a novel cell entry pathway. Gene Therapy 2005; 12: 225–237.

    Article  CAS  PubMed  Google Scholar 

  9. Chiu CY, Mathias P, Nemerow GR, Stewart PL . Structure of adenovirus complexed with its internalization receptor, alphavbeta5 integrin. J Virol 1999; 73: 6759–6768.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Miyamoto S, Akiyama SK, Yamada KM . Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 1995; 267: 883–885.

    Article  CAS  PubMed  Google Scholar 

  11. Wang K, Huang S, Kapoor-Munshi A, Nemerow G . Adenovirus internalization and infection require dynamin. J Virol 1998; 72: 3455–3458.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Seth P, Willingham MC, Pastan I . Binding of adenovirus and its external proteins to Triton X-114. Dependence on pH. J Biol Chem 1985; 260: 14431–14434.

    CAS  PubMed  Google Scholar 

  13. Leopold PL, Kreitzer G, Miyazawa N, Rempel S, Pfister KK, Rodriguez-Boulan E et al. Dynein- and microtubule-mediated translocation of adenovirus serotype 5 occurs after endosomal lysis. Hum Gene Ther 2000; 11: 151–165.

    Article  CAS  PubMed  Google Scholar 

  14. Suomalainen M, Nakano MY, Keller S, Boucke K, Stidwill RP, Greber UF . Microtubule-dependent plus- and minus end-directed motilities are competing processes for nuclear targeting of adenovirus. J Cell Biol 1999; 144: 657–672.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Seth P, Fitzgerald D, Ginsberg H, Willingham M, Pastan I . Evidence that the penton base of adenovirus is involved in potentiation of toxicity of Pseudomonas exotoxin conjugated to epidermal growth factor. Mol Cell Biol 1984; 4: 1528–1533.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Medina-Kauwe LK, Kasahara N, Kedes L . 3PO, a novel non-viral gene delivery system using engineered Ad5 penton proteins. Gene Therapy 2001; 8: 795–803.

    Article  CAS  PubMed  Google Scholar 

  17. Fender P, Ruigrok RW, Gout E, Buffet S, Chroboczek J . Adenovirus dodecahedron, a new vector for human gene transfer [see comments]. Nat Biotechnol 1997; 15: 52–56.

    Article  CAS  PubMed  Google Scholar 

  18. Bal HP, Chroboczek J, Schoehn G, Ruigrok RW, Dewhurst S . Adenovirus type 7 penton purification of soluble pentamers from Escherichia coli and development of an integrin-dependent gene delivery system. Eur J Biochem 2000; 267: 6074–6081.

    Article  CAS  PubMed  Google Scholar 

  19. Evanko SP, Parks WT, Wight TN . Intracellular hyaluronan in arterial smooth muscle cells: association with microtubules, RHAMM, and the mitotic spindle. J Histochem Cytochem 2004; 52: 1525–1535.

    Article  CAS  PubMed  Google Scholar 

  20. Lin SX, Collins CA . Immunolocalization of cytoplasmic dynein to lysosomes in cultured cells. J Cell Sci 1992; 101: 125–137.

    CAS  PubMed  Google Scholar 

  21. Bailey CJ, Crystal RG, Leopold PL . Association of adenovirus with the microtubule organizing center. J Virol 2003; 77: 13275–13287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Burkhardt JK, Echeverri CJ, Nilsson T, Vallee RB . Overexpression of the dynamitin (p50) subunit of the dynactin complex disrupts dynein-dependent maintenance of membrane organelle distribution. J Cell Biol 1997; 139: 469–484.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Greber UF, Willetts M, Webster P, Helenius A . Stepwise dismantling of adenovirus 2 during entry into cells. Cell 1993; 75: 477–486.

    Article  CAS  PubMed  Google Scholar 

  24. Varga MJ, Weibull C, Everitt E . Infectious entry pathway of adenovirus type 2. J Virol 1991; 65: 6061–6070.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Mukherjee S, Ghosh RN, Maxfield FR . Endocytosis. Physiol Rev 1997; 77: 759–803.

    Article  CAS  PubMed  Google Scholar 

  26. Kirchhausen T . Adaptors for clathrin-mediated traffic. Annu Rev Cell Dev Biol 1999; 15: 705–732.

    Article  CAS  PubMed  Google Scholar 

  27. Heilker R, Spiess M, Crottet P . Recognition of sorting signals by clathrin adaptors. BioEssays 1999; 21: 558–567.

    Article  CAS  PubMed  Google Scholar 

  28. Lencer WI, Hirst TR, Holmes RK . Membrane traffic and the cellular uptake of cholera toxin. Biochim Biophys Acta 1999; 1450: 177–190.

    Article  CAS  PubMed  Google Scholar 

  29. Pelkmans L, Kartenbeck J, Helenius A . Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 2001; 3: 473–483.

    Article  CAS  PubMed  Google Scholar 

  30. Norkin LC, Anderson HA, Wolfrom SA, Oppenheim A . Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles. J Virol 2002; 76: 5156–5166.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mundy DI, Machleidt T, Ying YS, Anderson RG, Bloom GS . Dual control of caveolar membrane traffic by microtubules and the actin cytoskeleton. J Cell Sci 2002; 115: 4327–4339.

    Article  CAS  PubMed  Google Scholar 

  32. Lisanti MP, Tang ZL, Sargiacomo M . Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae. J Cell Biol 1993; 123: 595–604.

    Article  CAS  PubMed  Google Scholar 

  33. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, Anderson RG . Caveolin, a protein component of caveolae membrane coats. Cell 1992; 68: 673–682.

    Article  CAS  PubMed  Google Scholar 

  34. Dangoria NS, Breau WC, Anderson HA, Cishek DM, Norkin LC . Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry. J Gen Virol 1996; 77: 2173–2182.

    Article  CAS  PubMed  Google Scholar 

  35. Rejman J, Oberle V, Zuhorn IS, Hoekstra D . Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 2004; 377: 159–169.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Lencer WI, Tsai B . The intracellular voyage of cholera toxin: going retro. Trends Biochem Sci 2003; 28: 639–645.

    Article  CAS  PubMed  Google Scholar 

  37. Lord JM, Deeks E, Marsden CJ, Moore K, Pateman C, Smith DC et al. Retrograde transport of toxins across the endoplasmic reticulum membrane. Biochem Soc Trans 2003; 31: 1260–1262.

    Article  CAS  PubMed  Google Scholar 

  38. Orlandi PA, Curran PK, Fishman PH . Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action. J Biol Chem 1993; 268: 12010–12016.

    CAS  PubMed  Google Scholar 

  39. Ros-Baro A, Lopez-Iglesias C, Peiro S, Bellido D, Palacin M, Zorzano A et al. Lipid rafts are required for GLUT4 internalization in adipose cells. Proc Natl Acad Sci USA 2001; 98: 12050–12055 [E-pub 12001 Oct 12052].

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Subtil A, Gaidarov I, Kobylarz K, Lampson MA, Keen JH, McGraw TE . Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc Natl Acad Sci USA 1999; 96: 6775–6780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Salone B, Martina Y, Piersanti S, Cundari E, Cherubini G, Franqueville L et al. Integrin alpha3beta1 is an alternative cellular receptor for adenovirus serotype 5. J Virol 2003; 77: 13448–13454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hong SS, Gay B, Karayan L, Dabauvalle MC, Boulanger P . Cellular uptake and nuclear delivery of recombinant adenovirus penton base. Virology 1999; 262: 163–177.

    Article  CAS  PubMed  Google Scholar 

  43. Bantel-Schaal U, Hub B, Kartenbeck J . Endocytosis of adeno-associated virus type 5 leads to accumulation of virus particles in the Golgi compartment. J Virol 2002; 76: 2340–2349.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Sandvig K, van Deurs B . Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. EMBO J 2000; 19: 5943–5950.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Carver LA, Schnitzer JE . Caveolae: mining little caves for new cancer targets. Nat Rev Cancer 2003; 3: 571–581.

    Article  CAS  PubMed  Google Scholar 

  46. Liu S, Calderwood DA, Ginsberg MH . Integrin cytoplasmic domain-binding proteins. J Cell Sci 2000; 113: 3563–3571.

    CAS  PubMed  Google Scholar 

  47. Marjomaki V, Pietiainen V, Matilainen H, Upla P, Ivaska J, Nissinen L et al. Internalization of echovirus 1 in caveolae. J Virol 2002; 76: 1856–1865.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chun J, Hyun S, Kwon T, Lee EJ, Hong SK, Kang SS . The subcellular localization control of integrin linked kinase 1 through its protein–protein interaction with caveolin-1. Cell Signal 2005; 17: 751–760. Epub 2004 Nov 2023.

    Article  CAS  PubMed  Google Scholar 

  49. Querbes W, Benmerah A, Tosoni D, Di Fiore PP, Atwood WJ . A JC virus-induced signal is required for infection of glial cells by a clathrin- and eps15-dependent pathway. J Virol 2004; 78: 250–256.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Meier O, Boucke K, Hammer SV, Keller S, Stidwill RP, Hemmi S et al. Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. J Cell Biol 2002; 158: 1119–1131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Miyazawa N, Leopold PL, Hackett NR, Ferris B, Worgall S, Falck-Pedersen E et al. Fiber swap between adenovirus subgroups B and C alters intracellular trafficking of adenovirus gene transfer vectors. J Virol 1999; 73: 6056–6065.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Miyazawa N, Crystal RG, Leopold PL . Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein. J Virol 2001; 75: 1387–1400.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kim D-H, Longo M, Han Y, Lundberg P, Cantin E, Rossi JJ . Interferon induction by siRNAs and ssRNAs synthesized by phage polymerase. Nat Biotechnol 2004; 22: 321–325.

    Article  CAS  PubMed  Google Scholar 

  54. Wiethoff CM, Wodrich H, Gerace L, Nemerow GR . Adenovirus protein VI mediates membrane disruption following capsid disassembly. J Virol 2005; 79: 1992–2000.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lukacs GL, Haggie P, Seksek O, Lechardeur D, Freedman N, Verkman AS . Size-dependent DNA mobility in cytoplasm and nucleus. J Biol Chem 2000; 275: 1625–1629.

    Article  CAS  PubMed  Google Scholar 

  56. Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene Therapy 1999; 6: 482–497.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the following people for helpful discussions on this work: Raj Batra, Maria Castro, Xinhua Chen, Nori Kasahara, Larry Kedes, Pedro Lowenstein and Kolja Wawrowsky. This work was supported by grants to LKM-K from the National Institutes of Health (CA102126), the Susan G Komen Breast Cancer Foundation (BCTR02-1194), the American Cancer Society (IRG individual allocation) and the Donna and Jesse Garber award. SHA and JX were supported by EY-13949 from the National Eye Institute. SHA was additionally supported by EY-11386, EY-05081, DK-56040 and GM-59297. MM was supported by NIH P30 DK48522 (Confocal Microscopy Subcore, USC Center for Liver Diseases).

Author information

Authors and Affiliations

  1. Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    A Rentsendorj, H Agadjanian, S Bass & L K Medina-Kauwe

  2. Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA

    J Xie, M MacVeigh & S F Hamm-Alvarez

  3. The Beckman Research Institute, City of Hope, Duarte, CA, USA

    D-H Kim & J Rossi

  4. Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    L K Medina-Kauwe

Authors
  1. A Rentsendorj
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M MacVeigh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H Agadjanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S Bass
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. D-H Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. J Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. S F Hamm-Alvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. L K Medina-Kauwe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to L K Medina-Kauwe.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rentsendorj, A., Xie, J., MacVeigh, M. et al. Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer. Gene Ther 13, 821–836 (2006). https://doi.org/10.1038/sj.gt.3302729

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302729

Keywords

This article is cited by

Search

Advanced search

Quick links