Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of ␣3␤1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin ␣3␤1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-␣3 and anti-␤1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with ␣3␤1, which involved multiple additional contact sites.Adenovirus (Ad) host cell entry requires an initial attachment to cells which is mediated by the fiber interaction with the coxsackievirus-Ad receptor (CAR) (2). The subsequent association of the capsid protein penton base with integrin molecules promotes Ad entry (31). Integrins are a family of structurally and functionally related cell surface heterodimeric receptors that mediate cell migration and adhesion. The major extracellular ligands for integrins are collagens, laminins, fibronectin, tenascin, vitronectin, von Willebrand factor, and fibrinogen, reflecting the primary function of integrins in cell adhesion to the extracellular matrix. The ␣v␤1, -3, -5, -6, and -8 integrins, the ␣5␤1 and ␣8␤1 integrins, and the ␣IIb␤3 integrins form a subgroup that primarily recognizes ligands containing Arg-Gly-Asp (RGD) motifs (see reference 13 and references therein). Many microorganisms utilize integrins to gain entry into cells: the SA11 rotavirus binds to ␣2␤1 and ␣4␤1 (9), ␣v␤3 and ␣v␤1 integrins are receptors of the human parechovirus 1 (30), and ␣v␤5 has been proposed, although not conclusively, as a coreceptor in adeno-associated virus type 2 infection (27, 29). The foot-and-mouth disease virus uses different integrins for cell infection (14,15,16). Integrin ␣3␤1 is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (1). Yersinia pseudotuberculosis binds to members of the ␤1 integrin family in order to enter eukaryotic cells (22).Several Ad serotypes contain an RGD motif in the penton base protein. This feature, and the Ad cell-detaching property, suggested an interaction of the virus with the integrin receptors. Indeed, ␣v␤3 and ␣v␤5 are receptors for human Ad2 and Ad5, and direct binding to isolated ␣v␤5 was shown for human Ad2, Ad3, Ad4, Ad5, and Ad37 (24, 31). In hematopoietic and melanoma cells, respectively, the ␣M␤2 and b1 integrins were found to be implicated in human Ad5 infection (3, 12). More recent evidence indicates ␣v␤1 as an Ad2 and Ad5 coreceptor in the human embryonic kidney (HEK293) cell line (23). Ad interaction with the ␣v␤1, -3, and -5 integrin subtypes is efficiently competed by RGD-containing peptides (23, 31). A second integrin binding motif is...

The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.

No abstract

No abstract

In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda 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 lambda 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 ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda 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 lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, and the related cytokine lymphotoxin α (LTα), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-α/LTα−/−), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-α/LTα−/− and TNF-α/LTα+/− mice versus TNF-α/LTα+/+ mice links antibody levels to TNF-α/LTα gene dosage. Due to the absence of neutralizing antibodies, the TNF-α/LTα knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-α in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.

No abstract

No abstract