Journal of Molecular Medicine

, Volume 82, Issue 7, pp 467–476 | Cite as

Mammalian cell transduction and internalization properties of λ phages displaying the full-length adenoviral penton base or its central domain

  • Stefania Piersanti
  • Gioia Cherubini
  • Yuri Martina
  • Barbara Salone
  • Daniele Avitabile
  • Fabiana Grosso
  • Enrico Cundari
  • Giovanni Di Zenzo
  • Isabella Saggio
Original Article


In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage λ is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered λ by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286–393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286–393) includes the principal receptor-binding motif (340RGD342). Both the phage chimerae λ Pb and λ Pb (286–393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of λ Pb and λ Pb (286–393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of λ Pb (286–393), but not that of λ Pb. The proteasome inhibitor MG-132 improved the efficiency of λ Pb (286–393)-mediated transduction, but not that of λ Pb. In summary, this work shows the feasibility of using λ phage as an efficient vector for gene transfer into mammalian cells. We show that λ Pb and λ Pb (286–393) can both mediate receptor-dependent transduction; while only λ Pb is able to promote endosomal escape and proteasome resistance of phage particles.


Bacteriophage Adenovirus Gene therapy Endosomal escape 



This work was supported by contributions from CNR, Progetto Finalizzato Biotecnologie, Istituto Pasteur Cenci Bolognetti, Università di Roma La Sapienza, MIUR, and Consorzio Interuniversitario Biotecnologie. We are grateful to P. Boulanger, A. Orecchia, and M. Nakanishi for their gifts of materials. We thank G. Ragone, F. Nasorri, M.C. Bonaccorsi, V. Velotta and P. Orlando for technical assistance, and L. Cordier, P. Bianco and C.M. Failla for fruitful scientific discussion.


  1. 1.
    Thomas C, Ehrhardt A, Kay M (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4:346–358CrossRefPubMedGoogle Scholar
  2. 2.
    Chen D, Murphy B, Sung R, Bromberg I (2003) Adaptive and innate immune responses to gene transfer vectors: role of cytokines and chemokines in vector function. Gene Ther 10:991–998CrossRefPubMedGoogle Scholar
  3. 3.
    Herweijer H, Wolff J (2003) Progress and prospects: naked DNA gene transfer and therapy. Gene Ther 10:453–458CrossRefPubMedGoogle Scholar
  4. 4.
    Schmidt-Wolf G, Schmidt-Wolf I (2003) Non-viral and hybrid vectors in human gene therapy: an update. Trends Mol Med 9:67–72Google Scholar
  5. 5.
    Di Giovine M, Salone B, Martina Y, Amati V, Zambruno G, Cundari E, Failla C, Saggio I (2001) Binding properties, cell delivery and gene transfer of adenoviral penton base-displaying bacteriophage. Virology 282:102–112CrossRefPubMedGoogle Scholar
  6. 6.
    Hart S, Knight A, Harbottle R, Mistry A, Hunger H, Cutler D, Williamson R, Coutelle C (1994) Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp containing peptide. J Biol Chem 269:12468–12474PubMedGoogle Scholar
  7. 7.
    Larocca D, Witte A, Johnson W, Pierce GF, Baird A (1998) Targeting bacteriophage to mammalian cell surface receptors for gene delivery. Hum Gene Ther 9:2393–2399PubMedGoogle Scholar
  8. 8.
    Larocca D, Kassner P, Witte A, Ladner R, Pierce G, Baird A (1999) Gene transfer to mammalian cells using genetically targeted filamentous bacteriophages. FASEB J 6:727–734Google Scholar
  9. 9.
    Larocca D, Jensen-Pergakes K, Burg M, Baird A (2001) Receptor-targeted gene delivery using multivalent phagemid particles. Mol Ther 3:476–484CrossRefPubMedGoogle Scholar
  10. 10.
    Poul M, Marks J (1999) Targeted gene delivery to mammalian cells by filamentous phage. J Mol Biol 288:203–211CrossRefPubMedGoogle Scholar
  11. 11.
    Felici F, Luzzago A, Monaci P, Nicosia A, Sollazzo M, Traboni C (1995) Peptide and protein display on the surface of filamentous bacteriophages. Biotechnol Annu Rev 1:149–183PubMedGoogle Scholar
  12. 12.
    Hufton S, Moerkerk P, Meulemans E, de Bruine A, Arends J, Hoogenboom H (1999) Phage display of cDNA repertoires: the pVI display system and its applications for the selection of immunogenic ligands. J Immunol Methods 231:39–51CrossRefPubMedGoogle Scholar
  13. 13.
    Maruyama IN, Brenner S (1992) A selective lambda phage cloning vector with automatic excision of the insert in a plasmid. Gene 120:135–141CrossRefPubMedGoogle Scholar
  14. 14.
    Parks RJ, Bramson JL, Wan Y, Addison CL, Graham FL (1999) Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors. J Virol 73:8027–8034PubMedGoogle Scholar
  15. 15.
    Heller H, Kammer C, Wilgenbus P, Doerfler W (1995) Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments. Proc Natl Acad Sci USA 92:5515–5519Google Scholar
  16. 16.
    Kessner P, Burg M, Baird A, Larocca D (1999) Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells. Bioch Biophys Res Comm 264:921–928CrossRefGoogle Scholar
  17. 17.
    Eguchi A, Akuta H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M (2001) Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J Biol Chem 276:26204–26210CrossRefPubMedGoogle Scholar
  18. 18.
    Dunn I (1995) Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein. J Mol Biol 248:497–506CrossRefPubMedGoogle Scholar
  19. 19.
    Mikawa Y, Maruyama I, Brenner S (1996) Surface display of proteins on bacteriophage lambda heads. J Mol Biol 262:21–30CrossRefPubMedGoogle Scholar
  20. 20.
    Santi E, Capone S, Mennuni C, Lahm A, Tramontano A, Luzzago A, Nicosia A (2000) Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics. J Mol Biol 296:497–508CrossRefPubMedGoogle Scholar
  21. 21.
    Hong S, Gay B, Karayan L, Dabauvalle M, Boulanger P (1999) Cellular uptake and nuclear delivery of recombinant adenovirus penton base. Virology 262:163–177CrossRefPubMedGoogle Scholar
  22. 22.
    Wickham TJ, Mathias P, Cheresh DA, Nemerow GR (1993) Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment. Cell 73:309–319PubMedGoogle Scholar
  23. 23.
    Li E, Brown S, Stupack D, Puente X, Cheresh D, Nemerow G (2001) Integrin αvβ1 is an adenovirus coreceptor. J Virol 75:5405–5409CrossRefPubMedGoogle Scholar
  24. 24.
    Salone B, Martina Y, Piersanti S, Cundari E, Cherubini G, Franqueville L, Failla C, Saggio I (2003) Integrin alpha3beta1 is an alternative receptor for adenovirus serotype 5. J Virol 77:13448–13454CrossRefPubMedGoogle Scholar
  25. 25.
    Stratford-Perricaudet LD, Makeh I, Perricaudet M, Briand P (1992) Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest 90:626–630PubMedGoogle Scholar
  26. 26.
    Sternberg N, Hoess R (1995) Display of peptides and proteins on the surface of bacteriophage λ. Proc Natl Acad Sci USA 92:1609–1613PubMedGoogle Scholar
  27. 27.
    Klein D (2002) Quantification using real-time PCR technology: applications and limitations. Trends Mol Med 8:257–260Google Scholar
  28. 28.
    Piazza C, Gilardini-Montani MS, Moretti S, Cundari E, Piccolella E (1997) Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis. J Immunol 158:1503–1506PubMedGoogle Scholar
  29. 29.
    Ruoslathi E (1996) RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12:697–715CrossRefPubMedGoogle Scholar
  30. 30.
    Bayer N, Schober D, Prchla E, Murphy R, Blaas D, Fuchs R (1998) Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection. J Virol 72:9645–9655PubMedGoogle Scholar
  31. 31.
    Jensen TJ, Loo MA, Pind S, Williams DB, Goldberg AL, Riordan JR (1995) Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83:129–135PubMedGoogle Scholar
  32. 32.
    Douar A, Poulard K, Stockholm D, Danos O (2001) Intracellular trafficking of adeno-associated virus vectors: routing to late endosomal compartment and proteasome degradation. J Virol 75:1824–1833CrossRefPubMedGoogle Scholar
  33. 33.
    Burg M, Jensen-Pergakes K, Gonzalez A, Ravey P, Baird A, Larocca D (2002) Enhanced phagemid particle gene transfer in camptothecin-treated carcinoma cells. Cancer Res 62:977–981PubMedGoogle Scholar
  34. 34.
    Karayan L, Hong SS, Gay B, Tournier J, Angeac ADD, Boulanger P (1997) Structural and functional determinants in adenovirus type 2 penton base recombinant protein. J Virol 71:8678–8679PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Stefania Piersanti
    • 1
  • Gioia Cherubini
    • 1
  • Yuri Martina
    • 1
  • Barbara Salone
    • 1
  • Daniele Avitabile
    • 1
  • Fabiana Grosso
    • 2
  • Enrico Cundari
    • 3
  • Giovanni Di Zenzo
    • 2
  • Isabella Saggio
    • 1
    • 3
  1. 1.Department of Genetics and Molecular BiologyUniversity La Sapienza and Parco Scientifico Biomedico di RomaRomeItaly
  2. 2.Laboratory of Molecular and Cell BiologyIDI-IRCCSRomeItaly
  3. 3.Institute of Cell Biology and PathologyCNRRomeItaly

Personalised recommendations