This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mathias, P.
Right arrow Articles by Nemerow, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mathias, P.
Right arrow Articles by Nemerow, G. R.

 Previous Article  |  Next Article 

Journal of Virology, November 1998, p. 8669-8675, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Interactions of Soluble Recombinant Integrin alpha vbeta 5 with Human Adenoviruses

Patricia Mathias,1 Michael Galleno,2 and Glen R. Nemerow1,*

The Scripps Research Institute, La Jolla,1 and Invitrogen, Carlsbad,2 California

Received 11 June 1998/Accepted 7 August 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha v integrins have been identified as coreceptors for adenovirus (Ad) internalization; however, direct interactions of these molecules with Ad have not been demonstrated. We report here the expression of soluble integrin alpha vbeta 5, which retains the ability to recognize the Ad penton base as well as vitronectin, an Arg Gly Asp (RGD)-containing extracellular matrix protein. Soluble integrin alpha vbeta 5 reacted with seven different Ad serotypes (subgroups A to E) in solid-phase binding assays. The soluble integrin exhibited different levels of binding to each Ad serotype; however, binding to multiple Ad types required the presence of divalent metal cations and was inhibited by a synthetic RGD peptide, indicating that RGD and cation-binding sequences regulate Ad interactions with alpha vbeta 5. Incubation of Ad particles with soluble alpha vbeta 5 integrin also inhibited subsequent Ad internalization into epithelial cells as well as virus attachment to monocytic cells. These findings suggest that soluble alpha v integrins or antagonists of these coreceptors could be used to limit infection by multiple Ad types. The generation of soluble alpha v integrins should also permit further detailed kinetic and structural analysis of Ad interactions with its coreceptors.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Integrins are a large family of heterodimeric receptors which mediate several important cell functions, including cell adhesion, cell growth and differentiation, cell motility, wound repair (16), and phagocytosis (29). Inappropriate or reduced expression of these receptors on certain cell types in vivo is also associated with the development of tumor growth and metastasis (9) and retinal degeneration (10). The pairing of different alpha  and beta integrins subunits determines the ligand binding specificity of each receptor (20). Several different integrins recognize the Arg Gly Asp (RGD) sequence in different extracellular matrix proteins, such as fibronectin and vitronectin (28), and also require the presence of divalent metal cations for binding (21).

Integrins have also been subverted by a number of different viral (5, 22, 27, 34, 36) and bacterial (6, 17, 18) pathogens in order to gain entrance to host cells. In the majority of cases, integrins mediate the initial attachment of the pathogen to the host cell surface. However, human adenoviruses (Ad) use the vitronectin binding integrins alpha vbeta 3 and alpha vbeta 5 to promote virus internalization (3, 36) rather than virus attachment (4, 33). Ad entry into hematopoietic cells is mediated by distinct integrin receptors that facilitate both virus attachment (alpha Mbeta 2) and internalization (alpha vbeta 3, alpha vbeta 5) (14).

alpha v integrins recognize an RGD sequence on the Ad penton base, which is conserved among several different virus serotypes (23). Recent structural studies have localized the alpha v integrin binding site on intact Ad serotype 2 (Ad2) particles by using cryoelectron microscopy and image reconstruction (31). The integrin binding sites, identified by the use of an RGD-specific monoclonal antibody (MAb) (designated DAV-1), are located at the apex of exposed, highly mobile loops on the Ad2 penton base protein. More recent structural studies have revealed that different Ad serotypes contain different-size RGD protrusions on the penton base protein (5a); however, it is not yet known how this influences integrin binding.

While integrins alpha vbeta 3 and alpha vbeta 5 both support Ad internalization, integrin alpha vbeta 5 selectively plays an important role in subsequent steps in virus penetration (36). Binding of the penton base protein to integrin alpha vbeta 5 at reduced pH promotes Ad permeabilization of the cell membrane. Integrin alpha vbeta 5 is expressed on human bronchial epithelial cells (25), a major site of Ad infection in vivo. In contrast, primary epithelial cells express little if any integrin alpha vbeta 3. The level of alpha vbeta 5 integrin expression on human airway epithelial cells in vivo has also been shown to be directly related to the efficiency of Ad-mediated gene transfer (11, 12).

In order to gain a better understanding of Ad interactions with its integrin coreceptors, we have produced the entire ectodomain of integrin alpha vbeta 5 as a secreted protein in insect cells using baculovirus vectors. Direct interactions of the secreted integrin with multiple Ad serotypes, as well as host cell-derived RGD ligands, were examined by solid-phase binding assays.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Viruses, extracellular matrix proteins, and antibodies. Ad2, Ad3, Ad4, Ad12, Ad19, and Ad37 were purchased from the American Type Culture Collection (Manassas, Va.) and propagated in A549 cells. A recombinant Ad5 vector expressing green fluorescent protein (Ad.GFP) was grown in 293 cells (15). Viruses were purified by CsCl density gradient ultracentrifugation, as previously described (8). For virus binding and internalization assays, purified Ad2 was labeled with 125NaI (IODO-GEN; Pierce) to a specific activity of 3.5 × 104 cpm/ng. Recombinant Ad2 penton base was produced in insect cells by using baculovirus, as previously described (36). Flockhouse virus (FHV), a nonenveloped insect virus, was kindly provided by A. Schneemann (Scripps Research Institute). Extracellular matrix proteins vitronectin and fibronectin were purchased from Sigma (St. Louis, Mo.). A non-function-blocking anti-alpha v MAb (LM142) and a function-blocking anti-alpha vbeta 5 MAb (P1F6) were kindly provided by D. Cheresh (Scripps Research Institute). MAbs directed against the alpha v subunit (VNR139; Chemicon), the beta 5 subunit (1D11;D. Cheresh), or alpha Mbeta 2 (CP3; Z. Ruggeri, Scripps Research Institute) were used for immunoblotting.

Construction of baculovirus vectors encoding soluble alpha vFos and beta 5Jun integrin subunits. A 2,941-bp cDNA encoding the entire alpha v integrin ectodomain (32) was PCR amplified (Pfu polymerase; Stratagene) from a plasmid [pGEM7zf(+)] encoding the full-length integrin subunit (kindly provided by D. Cheresh). An appropriate set of PCR primers (5'-GCGCGGCTAGCGACGATGGCTT, 3'-CATGGTACCTGGCTGAATGCCC) was used to create NheI and KpnI restriction endonuclease cloning sites (underlined). Following sequencing, the 2.9-kb cDNA fragment was digested with NheI and KpnI and ligated into the baculovirus transfer vector pBB4 (Invitrogen), using the same cloning sites. The 2.9-kb alpha v cDNA, as well as a 5' KpnI and a 3' stop codon and EcoRI cloning sites, was also inserted into pBB4 in frame with a 144-bp cDNA fragment encoding the Fos dimerization domain (pPIC9DraFOS) (19).

Using a similar cloning strategy, we PCR amplified a 2,154-bp cDNA fragment encoding the entire ectodomain of the beta 5 integrin subunit (26) (pCI/beta 5; D. Cheresh) with BamHI and XhoI restriction cloning sites and PCR primers (5'-AGGGGATCCACCATGCCGCGGG, 3'-ATGGTCTCGAGGTTGGGGGTGTT). Following sequencing, the 2.1-kb fragment was digested with BamHI and XhoI and ligated into the pBB4 baculovirus transfer vector with the same cloning sites. A separate construct was also generated to produce beta 5 linked to the Jun dimerization domain. pBB4/beta 5 was digested with XhoI and EcoRI, and a 144-bp fragment containing a polyglycine linker sequence and the Jun dimerization domain (pPIC9DRbeta Jun) was digested with SalI and EcoRI and then ligated in frame with the truncated beta 5 cDNA in pBB4, which had been previously digested with XhoI and EcoRI.

To generate recombinant baculoviruses, Sf9 insect cells were cotransfected with 0.5 µg of wild-type baculovirus DNA (Bac'n Blue DNA; Invitrogen) and 4 µg of either pBB4/alpha v or pBB4/beta 5 or the same vectors containing the Fos and Jun forms of the integrin subunits (see Fig. 1). Following transfections, recombinant baculoviruses were isolated by plaque formation on Sf9 cells. Candidate recombinant baculoviruses were then amplified by growth on Sf9 cells, and viral DNA was isolated and analyzed on a 0.8% agarose gel and subsequently sequenced (Baculo/PCR; Invitrogen). High-titered baculovirus stocks expressing alpha v and beta 5 were generated by infection of Sf9 spinner cultures at a multiplicity of infection (MOI) of 0.3.

Immunoprecipitation, SDS-PAGE, and immunoblotting. For immunoprecipitation studies, 50% confluent monolayers of Tn5B cells in 24-well plastic tissue culture plates were infected with various ratios of alpha v and beta 5 recombinant baculoviruses or with each baculovirus alone. Eighteen hours postinfection, the cells were cultured for an additional 24 h in serum-free insect cell medium (Ex-Cell 400; JRH Biosciences, Lenexa, Kans.) containing 50 µCi of [35S]methionine (Amersham, Bedford, Mass.) per ml. The supernatants from infected or uninfected control cells were then harvested and incubated for 90 min at 4°C with 50 µl of protein G-Sepharose beads (Pierce) containing 10 µg of MAb P1F6. After a wash, the beads were boiled for 2 min in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and electrophoresed under nonreducing conditions on a 7% SDS gel (Tris-glycine; Novex). Following electrophoresis, the gel was fixed, dried, and subjected to autoradiography. For immunoblotting, soluble integrin was electrophoresed on an 8 to 16% SDS gel and then stained with Colloidal Coomassie blue (Novex) or transferred to a polyvinylidene difluoride membrane (Immobilon P; Amersham). The membrane was blocked by incubation in 10% nonfat dry milk in TBST (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.2% Tween 20). The blot was then probed with MAbs specific for the alpha v subunit (VNR 139) or the beta 5 subunit (1D11) or with a control anti-alpha Mbeta 2 antibody (CP3). Following extensive washing in TBST, the blots were incubated with a goat anti-mouse immunoglobulin G antibody linked to horseradish peroxidase (Bio-Rad). After further washes, the blots were developed with an enhanced chemiluminescence system (SuperSignal; Pierce).

Purification and ligand binding properties of soluble alpha vbeta 5 integrin. Tn5B insect cells were grown to a density of 2 × 106 cells/ml in Ex-cell 400 medium in 2-liter shake flasks and then infected at an MOI of 1.0 with recombinant baculoviruses encoding alpha v and beta 5 (1:1 ratio). Protease inhibitors (1 µg of leupeptin per ml, 5 µg of aprotinin per ml) and a final concentration of 2% fetal calf serum were added at 16 h postinfection, and the culture supernatants were harvested by centrifugation usually at 48 h postinfection, while the cells retained 90 to 100% viability. Culture supernatants were concentrated approximately 15-fold (Ultralab 2 concentrator; Pall Filtron) with a 30K membrane filter (Omega) and then adjusted to a final concentration of 0.002% sodium azide with additional protease inhibitors. The concentrated culture supernatants were incubated overnight at 4°C with constant agitation with MAb P1F6 (22 mg) covalently linked to 5 ml of CNBr-Sepharose beads. The beads were then washed five times with 50 ml of 10 mM sodium phosphate buffer, pH 7.4, containing 0.6 M NaCl and 1 mM (each) MgCl2 and CaCl2. The integrin was then eluted with 10 mM Na-acetate, pH 3.1, containing 1 mM MgCl2 and CaCl2 and immediately neutralized with 1 M Tris-HCl, pH 8.1. The eluted integrin fractions were analyzed by SDS-PAGE and for binding to the Ad2 penton base in an enzyme-linked immunosorbent assay (ELISA), as described below. Fractions containing the 155- and 100-kDa integrin heterodimer were pooled and concentrated (C-100 microconcentrator; Amicon) to approximately 300 µg/ml and stored at -80°C.

Integrin binding to Ad serotypes and extracellular matrix proteins was quantitated in an ELISA. Purified Ad serotypes, recombinant Ad2 penton base, or extracellular matrix proteins were used to coat 96-well plastic tissue culture plates (Immulon-4; Dynatech) at a concentration of 1 µg of protein/well. Nonspecific binding sites were quenched by incubation with a blocking agent (Superblock; Pierce), and then cell culture supernatants containing soluble integrin or immunoaffinity-purified integrin at 100 ng to 1 µg/well were added to the plates and incubated at 22°C for 2 h. The wells were washed with phosphate-buffered saline containing a 1:50 dilution of Blocker Blotto (Pierce)-0.2% Tween 20 and then incubated for 1 h at 22°C with 100 µl of 10 µg of LM142 anti-alpha v MAb per ml in blocking buffer. After further washes, the wells were incubated for 1 h with 100 µl of a 1:10,000 dilution of goat anti-mouse immunoglobulin G linked to horseradish peroxidase (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) in blocking buffer. Following additional washing, the reactions were developed by addition of substrate (ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid]; Kirkegaard and Perry Laboratories) and analyzed at 405 nm in a SpectraMax 250 ELISA plate reader (Molecular Devices, Sunnyvale, Calif.).

Ad binding, internalization, and gene delivery assays. 125I-labeled Ad2 (125 ng, 500 virus particles per cell) was preincubated for 2 h at 4°C with 70 µg/ml of a polyclonal antibody to the Ad2 fiber protein or the DAV-1 anti-penton base MAb (31) or with purified alpha vFos/beta 5Jun integrin in 10 mM Tris-HCl buffer, pH 8.1, containing 1 mM (each) MgCl2 and CaCl2. The samples were then incubated for 1 h at 4°C with 1 × 106 SW480 epithelial cells or 2 × 106 THP-1 monocytic cells in 20 mM HEPES-buffered saline (HBS), pH 7.4, containing 2% bovine serum albumin and 1 mM (each) MgCl2 and CaCl2. The cells were then warmed to 37°C for 0 to 15 min and then washed three times in cold HBS binding buffer. Cell samples maintained at 4°C were then counted to determine the level of virus attachment or were incubated with medium containing trypsin-EDTA at 37°C for 15 min to determine the amount of endocytosed virus particles. The amount of nonspecific Ad binding was determined by incubating cells with a 100-fold excess of unlabeled virus particles.

For gene delivery studies, 50 ng of Ad.GFP (MOI, 500) was incubated with 300 ng of soluble integrin in HBS binding buffer or in buffer alone for 2 h at 4°C. The samples were then added to 4 × 105 SW480, A549, or THP-1 cells and incubated further for 1 h at 4°C. The cells were then warmed to 37°C for 15 min and washed and cultured in complete Dulbecco modified Eagle medium for 48 to 72 h at 37°C. GFP expression was then quantitated by flow cytometry (FACScan II), and the data were expressed as mean fluorescence intensities.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Baculovirus expression of soluble integrin alpha vbeta 5. In order to study direct interactions of integrins with human Ad and host cell ligands, we used recombinant baculovirus vectors to express a soluble form of alpha vbeta 5 in Tn5B insect cells. To facilitate assembly of the integrin heterodimer (19), we inserted a glycine linker and Fos/Jun dimerization domain in frame with the C terminus of the integrin ectodomains (Fig. 1). Baculovirus transfer vectors containing these constructs were used to produce recombinant baculoviruses in Sf9 cells. Plaque-purified recombinant baculoviruses were then tested for the ability to produce a soluble alpha vbeta 5 integrin heterodimer in Tn5B insect cells by immunoprecipitation. Two proteins, of 155 and 100 kDa, similar to the expected size of the integrin ectodomains, were immunoprecipitated by a function-blocking alpha vbeta 5 MAb (P1F6) from supernatants of cells infected with both the alpha vFos and beta 5Jun baculoviruses (Fig. 2). An infection ratio of 1:1 proved optimal for integrin expression (Fig. 2, lanes 1 to 3). As expected, the integrin heterodimer was not detected in culture supernatants from cells infected with a alpha vFos or beta 5Jun baculovirus alone (lanes 5, 6). The beta 5Jun, but not the alpha vFos, integrin subunit was immunoprecipitated by MAb P1F6 (lane 6), while the alpha vFos integrin subunit was capable of being immunoprecipitated by MAb LM142 (data not shown).


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 1.   Diagram of baculovirus plasmid vectors used for the expression of soluble alpha vbeta 5. The cDNA fragments encoding the entire ectodomains of the alpha v and beta 5 integrin subunits were inserted in frame with a glycine spacer and a Fos/Jun dimerization domain (as shown in the sequence alignment) into the pBlueBac4 baculovirus transfer vector.


View larger version (101K):
[in this window]
[in a new window]
  		FIG. 2.   Immunoprecipitation analysis of soluble recombinant alpha vbeta 5 integrin. 35S-labeled insect cells were infected with a 1:1 (lane 1), 10:1 (lane 2), or 1:10 (lane 3) ratio (PFU) of the alpha vFos and beta 5Jun baculoviruses, were left uninfected (lane 4), or were infected with alpha vFos (lane 5) or beta 5Jun (lane 6) baculovirus alone and then immunoprecipitated with MAb P1F6 and analyzed under nonreducing conditions on an SDS-7% polyacrylamide gel, followed by autoradiography.

Studies were next performed to assess the functional activity of soluble integrin molecules produced in insect cells. Culture supernatants derived from cells infected with a combination of alpha vFos and beta 5Jun baculoviruses, but not those from cells infected with the alpha vFos or beta 5Jun baculovirus alone, reacted with the penton base protein in an ELISA (Fig. 3). Optimal expression occurred at 3 to 4 days postinfection and declined thereafter. These findings suggest that a functionally active integrin heterodimer was secreted from cells coinfected with alpha v and beta 5 recombinant baculoviruses.


View larger version (22K):
[in this window]
[in a new window]
  		FIG. 3.   Time course of expression and functional activity of soluble alpha vbeta 5 integrin. Culture supernatants derived from insect cells infected with a combination of the alpha vFos and beta 5Jun baculoviruses (1:1) or from cells infected with each baculovirus alone were assayed in an ELISA at various times after infection for binding to immobilized penton base.

Purification and ligand binding analysis of soluble integrin alpha vbeta 5. In order to study further the ligand binding properties of the Ad coreceptor, we isolated the soluble integrin by immunoaffinity chromatography. As expected, the purified integrin was comprised of two subunits with relative mobilities of 155 and 100 kDa (Fig. 4). Each of the integrin subunits was also recognized by the appropriate anti-alpha v or anti-beta 5 MAb, as detected by immunoblotting (Fig. 4). A minor protein of approximately 35 kDa, derived from proteolytic cleavage of the beta 5 integrin subunit, was detected in some integrin preparations. Approximately 250 to 300 µg of soluble integrin per liter of Tn5B insect cells was isolated.


View larger version (90K):
[in this window]
[in a new window]
  		FIG. 4.   SDS-PAGE and Western blot of immunoaffinity-purified alpha vbeta 5 integrin. Five micrograms of isolated alpha vbeta 5 were electrophoresed on an SDS-8 to 16% polyacrylamide gel under nonreducing conditions and then stained with Coomassie blue (lane 1) or transferred to a polyvinyidene difluoride membrane and reacted with an anti-alpha v (lane 2), anti-beta 5 (lane 3), or anti-alpha M (lane 4) antibody in a Western blot.

Further studies examined the functional properties of the purified alpha vbeta 5 integrin. Purified alpha vbeta 5 bound to immobilized Ad2 penton base and virus particles as well as to vitronectin but did not recognize another RGD-containing extracellular matrix protein, fibronectin (Fig. 5). Binding to Ad2 and vitronectin was blocked by 20 mM EDTA, indicating that these interactions require the presence of divalent metal cations. These results indicate that soluble recombinant integrin alpha vbeta 5 possesses the ligand binding activity of the cell-associated integrin.


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5.   Interactions of soluble alpha vbeta 5 integrin with Ad ligands or extracellular matrix proteins. ELISA plates were coated with 1 µg per well of penton base (PB), Ad2 particles, vitronectin (VN), fibronectin (FN), or bovine serum albumin (BSA). Following quenching of nonspecific binding sites, the plates were incubated with 6 ng of purified alpha vbeta 5 integrin in the presence (stippled bars) or absence (solid bars) of 20 mM EDTA. Integrin binding was detected with a non-function-blocking MAb (LM142), as described in Materials and Methods.

Association of integrin alpha vbeta 5 with different Ad serotypes. The penton base proteins of multiple Ad serotypes contain conserved RGD sequences (23), suggesting that different Ad are capable of associating with cell integrins. Competition studies with synthetic RGD peptides or function-blocking integrin MAbs also suggested that multiple Ad serotypes use integrins for infection. However, direct interactions of integrins with different Ad serotypes have not yet been examined. We therefore analyzed the association of soluble recombinant alpha vbeta 5 with seven different Ad serotypes representing five major Ad subgroups (A to E). Soluble alpha vbeta 5 exhibited significant binding to Ad serotypes belonging to subgroups B (Ad3), C (Ad2, Ad5), D (Ad19, Ad37), and E (Ad4) but did not bind to a control, nonenveloped virus, FHV (Fig. 6A). Minimal binding of alpha vbeta 5 to Ad12 was observed, consistent with the relatively short RGD surface loop on this virus (23). Binding of the soluble integrin to different Ad required the presence of divalent metal cations and was also inhibited by the presence of a synthetic RGD peptide (Fig. 6B).


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6.   Binding of soluble alpha vbeta 5 integrin to different Ad serotypes. (A) Direct interactions of soluble integrin with different Ad serotypes from subgroups A to E were quantitated in an ELISA in the presence (stippled bars) or absence (solid bars) of 20 mM EDTA. (B) Binding of soluble integrin alpha vbeta 5 to Ad2 and Ad37 was measured in the presence or absence (control) of 100 µg of a synthetic RGD peptide (RGDpep) per ml or 20 mM EDTA.

Effect of soluble integrin alpha vbeta 5 on Ad binding and internalization. Previous studies had indicated that alpha v integrin-deficient epithelial cells do not support efficient Ad internalization (36) and infection (12), whereas cells expressing these receptors following transfection with alpha v integrin cDNA are rendered susceptible to infection, suggesting that these receptors play a crucial role in viral entry. In order to substantiate this hypothesis, we preincubated purified virions with antibodies to the Ad2 fiber or the penton base protein or with soluble alpha vbeta 5 and then assayed virus binding and entry into host cells. Ad2 binding to SW480 epithelial cells was completely inhibited by an antifiber antibody, whereas incubation of virus with the DAV-1 penton base MAb or soluble alpha vbeta 5 integrin had little effect on binding (Fig. 7A). In contrast, soluble integrin alpha vbeta 5, as well as MAb DAV-1, significantly reduced (approximately 65%) Ad2 internalization (Fig. 7B). These findings are consistent with a specific role of alpha v integrins in Ad internalization into epithelial cells rather than in Ad attachment. In contrast to epithelial cells, both binding and internalization of Ad to monocytic cells has been reported to be mediated by penton base interaction with cell integrins (14). We therefore examined whether soluble alpha vbeta 5 was capable of interfering with Ad interactions with THP-1 monocytic cells. Consistent with the previous report, incubation of Ad2 with soluble integrin alpha vbeta 5 blocked both virus attachment to and internalization into THP-1 monocytic cells (Fig. 7C).


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 7.   Effect of antifiber or anti-penton base antibodies or soluble integrin alpha vbeta 5 on Ad2 binding and internalization. 125I-labeled Ad2 particles were preincubated with a polyclonal antifiber antibody with the DAV-1 anti-penton base MAb, or with soluble alpha vbeta 5 integrin prior to measurement of virus binding (A) and internalization into SW480 epithelial cells (B) or THP-1 monocytic cells (C).

Inhibition of Ad-mediated gene delivery by soluble integrin alpha vbeta 5. Further studies were performed to determine whether soluble integrin alpha vbeta 5 was capable of inhibiting Ad infection. For these studies we used a recombinant Ad5 vector encoding GFP (15). Incubation of Ad5.GFP with soluble alpha vbeta 5 significantly inhibited virus-mediated gene delivery to both SW480 and A549 epithelial cells (approximately 50%) and nearly abolished delivery to THP-1 monocytic cells (Fig. 8). These results further indicate that alpha v integrins promote Ad-mediated gene delivery as well as virus internalization.


View larger version (27K):
[in this window]
[in a new window]
  		FIG. 8.   Effect of soluble integrin alpha vbeta 5 on Ad-mediated gene delivery. A recombinant Ad vector encoding GFP (Ad.GFP) was preincubated with medium alone or with a 20-fold excess (relative to the number of penton base RGD sites) of soluble integrin alpha vbeta 5 prior to infection of SW480 cells, A549 epithelial cells, or THP-1 monocytic cells. Gene delivery was measured by flow cytometry at 48 to 72 h postinfection and expressed as mean fluorescence intensity. The data are representative of four separate experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The inherent difficulties associated with isolating native alpha v integrins from human tissues (30) has prevented an extensive analysis of integrin structure and function. The extracellular domains of integrins alpha vbeta 6 (35) and alpha IIbbeta 3 (24) have been previously expressed in mammalian or insect cells as secreted proteins. In each case, the expressed alpha  and beta  integrin subunits formed a stable heterodimer in the absence of the transmembrane or cytoplasmic domains and also retained antigenic properties and ligand binding activity. We were therefore encouraged to examine whether integrin alpha vbeta 5 could also be expressed as a soluble protein, thereby facilitating analysis of its interactions with human Ad. Unfortunately, only small amounts of the alpha v and beta 5 ectodomains were produced by baculovirus vectors in Tn5B insect cells. However, the addition of a Fos/Jun dimerization domain (Fig. 1) increased production of the soluble alpha vbeta 5 integrin approximately fourfold (data not shown). This could be due to increased association of the integrin subunits, as has been reported for other multimeric cell surface receptors (19). The soluble alpha v and beta 5 integrin subunits formed a stable heterodimeric complex (Fig. 2), and the assembled integrin was also capable of binding to the Ad penton base protein (Fig. 3) as well as its natural RGD-containing ligand, vitronectin (Fig. 5). The secreted alpha vbeta 5 integrin was also recognized by the function-blocking P1F6 MAb, which enabled isolation of the receptor by immunoaffinity chromatography (Fig. 4).

Since distinct Ad serotypes have been shown to contain a conserved integrin-binding motif (RGD) in their penton base proteins, we examined whether soluble alpha vbeta 5 integrin was capable of recognizing different Ad serotypes. While the level of integrin binding to different Ad types varied (Fig. 6A), competition studies indicated that the RGD sequence as well as the presence of divalent metal cations were required for integrin interactions with multiple Ad serotypes (Fig. 6B). The least amount of integrin binding observed was to Ad12. It is worth noting that the Ad12 penton base contains a relatively short RGD loop (23) compared to other Ad serotypes, a structural feature which could reduce integrin binding (1, 7). Previous studies have also reported that the Ad12 penton base RGD motif is capable of mediating cell adhesion and virus infection (2). However, significant differences in Ad2- and Ad12-mediated cell adhesion and infection were noted, indicating that the integrin binding epitopes on these two proteins are not functionally equivalent. One possibility which could explain reduced interactions of Ad12 with alpha vbeta 5 in solid-phase binding assays is that the Ad12 penton base has a low intrinsic affinity for integrin alpha vbeta 5. Recent structural studies have indicated that a more extended flexible RGD loop may be important for receptor interactions (1, 7). In vivo, high-affinity binding of Ad12 via the fiber receptor (4, 33) could permit subsequent low-affinity interactions of the penton base with the alpha vbeta 5 integrin. Further kinetic studies are needed to determine the precise binding affinities and stoichiometries of alpha v integrin interactions with different Ad penton base proteins.

The generation of a soluble integrin alpha vbeta 5 molecule also provided an opportunity to examine the requirements for this coreceptor in Ad entry and infection of epithelial or monocytic cells. Incubation of Ad2 particles with purified integrin had little effect on virus binding to epithelial cells; however, substantial inhibition of virus attachment to monocytic cells was observed (Fig. 7). We interpret this result as the ability of soluble alpha vbeta 5 to compete for Ad2 binding to cell surface alpha Mbeta 2 integrins (14). The integrin also inhibited virus internalization into both cell types. These results are consistent with a previous report (14), suggesting that integrins play distinct roles in diverse cell types. Soluble integrin alpha vbeta 5 was also capable of blocking Ad-mediated gene delivery to both epithelial and monocytic cells (Fig. 8). The more pronounced inhibition of gene delivery to THP1 monocytic cells (Fig. 7B) is probably due to the fact that integrins on these cells mediate both virus attachment and virus internalization.

The findings reported here suggest that soluble integrins or specific antagonists of these molecules may represent an approach to the inhibition of infection by multiple Ad serotypes. To date, there are few antiviral agents capable of inhibiting Ad infections. Although Ad infections do not generally cause serious complications, fatal disseminated Ad infections have been noted with increasing frequency in immunocompromised and AIDS patients (13). Strategies based on preventing Ad interactions with its cellular coreceptor may provide an approach to restricting cell entry by distinct Ad serotypes.

    ACKNOWLEDGMENTS

We express our gratitude to Phoebe Stewart and Charles Chiu (UCLA School of Medicine) and members of the Cheresh laboratory (Scripps Research Institute) for helpful discussions and advice. We also thank Bonnie Bradt for technical support and Catalina Hope and Joan Gausepohl for preparation of the manuscript.

This work was supported by NIH grants EY11431 and HL54352.

    FOOTNOTES

* Corresponding author. Mailing address: Department of Immunology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (619) 784-8472. Fax: (619) 784-8472. E-mail: gnemerow{at}scripps.edu.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Akke, M., J. Liu, J. Cavanagh, H. P. Erickson, and A. G. Palmer, III. 1998. Pervasive conformational fluctuations on microsecond time scales in a fibronectin type III domain. Nat. Struct. Biol. 5:55-59[Medline].
2. Bai, M., L. Campisi, and P. Freimuth. 1994. Vitronectin receptor antibodies inhibit infection of HeLa and A549 cells by adenovirus type 12 but not by adenovirus type 2. J. Virol. 68:5925-5932[Abstract/Free Full Text].
3. Bai, M., B. Harfe, and P. Freimuth. 1993. Mutations that alter an Arg-Gly-Asp (RGD) sequence in the adenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flat cells. J. Virol. 67:5198-5205[Abstract/Free Full Text].
4. Bergelson, J. M., J. A. Cunningham, G. Droguett, E. A. Kurt-Jones, A. Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg. 1997. Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320-1323[Abstract/Free Full Text].
5. Bergelson, J. M., M. P. Shepley, B. M. C. Chan, M. E. Hemler, and R. W. Finberg. 1992. Identification of the integrin VLA-2 as a receptor for echovirus I. Science 255:1718-1720[Abstract/Free Full Text].
5a. Chiu, C., et al. Submitted for publication.
6. Coburn, J., S. W. Barthold, and J. M. Leong. 1994. Diverse Lyme disease spirochetes bind integrin alpha IIbbeta 3 on human platelets. Infect. Immun. 62:5559-5567[Abstract/Free Full Text].
7. Copie, V., Y. Tomita, S. K. Akiyama, S. Aota, K. M. Yamada, R. M. Venable, and R. W. Pastor. 1998. Solution structure and dynamics of linked cell attachment modules of mouse. J. Mol. Biol. 277:663-682[Medline].
8. Everitt, E., S. A. Meador, and A. S. Levine. 1977. Synthesis and processing of the precursor to the major core protein of adenovirus type 2. J. Virol. 21:199-214[Abstract/Free Full Text].
9. Felding-Habermann, B., B. M. Mueller, C. A. Romerdahl, and D. A. Cheresh. 1992. Involvement of integrin alpha v gene expression in human melanoma tumorigenicity. J. Clin. Investig. 89:2018-2022.
10. Finnemann, S. C., V. L. Bonilha, A. D. Marmorstein, and E. Rodriguez-Boulan. 1997. Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha vbeta 5 integrin for binding but not for internalization. Proc. Natl. Acad. Sci. USA 94:12932-12937[Abstract/Free Full Text].
11. Goldman, M., Q. Su, and J. M. Wilson. 1996. Gradient of RGD-dependent entry of adenoviral vector in nasal and intrapulmonary epithelia: implications for gene therapy of cystic fibrosis. Gene Ther. 3:811-818[Medline].
12. Goldman, M. J., and J. M. Wilson. 1995. Expression of alpha vbeta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J. Virol. 69:5951-5958[Abstract].
13. Hierholzer, J. C. 1992. Adenoviruses in the immunocompromised host. Clin. Microbiol. Rev. 5:262-274[Abstract/Free Full Text].
14. Huang, S., T. Kamata, Y. Takada, Z. M. Ruggeri, and G. R. Nemerow. 1996. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J. Virol. 70:4502-4508[Abstract].
15. Huang, S., D. G. Stupack, P. Mathias, Y. Wang, and G. Nemerow. 1997. Growth arrest of Epstein-Barr virus immortalized B lymphocytes by adenovirus-delivered ribozymes. Proc. Natl. Acad. Sci. USA 94:8156-8161[Abstract/Free Full Text].
16. Hynes, R. O. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11-25[Medline].
17. Isberg, R. R., and G. Tran Van Nhieu. 1995. The mechanism of phagocytic uptake promoted by invasin-integrin interaction. Trends Cell Biol. 120:120-124.
18. Ishibashi, Y., S. Claus, and D. A. Relman. 1994. Bordeltella pertussis filamentous hemagglutinin interacts with a leukocyte signal transduction complex and stimulates bacterial adherence to monocyte CR3 (CD11b/CD18). J. Exp. Med. 180:1225-1233[Abstract/Free Full Text].
19. Kalandadze, A., M. Galleno, L. Foncerrada, J. L. Strominger, and K. W. Wucherpfennig. 1996. Expression of recombinant HLA-DR2 molecules. J. Biol. Chem. 271:20156-20162[Abstract/Free Full Text].
20. Kühn, K., and J. Eble. 1994. The structural bases of integrin-ligand interactions. Trends Cell Biol. 4:256-261. [Medline]
21. Loftus, J. W., J. C. Smith, and M. H. Ginsberg. 1994. Integrin-mediated cell adhesion: the extracellular face. J. Biol. Chem. 269:25235-25238[Free Full Text].
22. Mason, P. W., E. Rieder, and B. Baxt. 1994. RGD sequence of foot-and-mouth disease virus is essential for infecting cells via the natural receptor but can be bypassed by an antibody-dependent enhancement pathway. Proc. Natl. Acad. Sci. USA 91:1932-1936[Abstract/Free Full Text].
23. Mathias, P., T. J. Wickham, M. Moore, and G. Nemerow. 1994. Multiple adenovirus serotypes use alpha v integrins for infection. J. Virol. 68:6811-6814[Abstract/Free Full Text].
24. McKay, B. S., D. S. Annis, S. Honda, D. Christie, and T. J. Kunicki. 1996. Molecular requirements for assembly and function of a minimized human integrin. J. Biol. Chem. 271:30544-30547[Abstract/Free Full Text].
25. Mette, S. A., J. Pilewski, C. A. Buck, and S. M. Albelda. 1993. Distribution of integrin cell adhesion receptors on normal bronchial epithelial cells and lung cancer cells in vitro and in vivo. Am. J. Respir. Cell Mol. Biol. 8:562-572.
26. Ramaswamy, H., and M. E. Hemler. 1990. Cloning, primary structure and properties of a novel human integrin beta  subunit. EMBO J. 9:1561-1568[Medline].
27. Roivaninen, M., T. Hypiä, L. Pirainen, N. Kalkkinen, G. Stanway, and T. Hovi. 1991. RGD-dependent entry of coxsackievirus A9 into host cells and its bypass after cleavage of VP1 protein by intestinal proteases. J. Virol. 65:4735-4740[Abstract/Free Full Text].
28. Ruoslahti, E., and M. D. Pierschbacher. 1987. New perspectives in cell adhesion: RGD and integrins. Science 238:491-497[Abstract/Free Full Text].
29. Savill, J., I. Dransfield, N. Hogg, and C. Haslett. 1990. Vitronectin receptor-mediated phagocytosis of cells undergoing apoptosis. Nature 343:170-173[Medline].
30. Smith, J. W., D. J. Vestal, S. V. Irwin, T. A. Burke, and D. A. Cheresh. 1990. Purification and functional characterization of integrin alpha vbeta 5: an adhesion receptor for vitronectin. J. Biol. Chem. 265:11008-11013[Abstract/Free Full Text].
31. Stewart, P. L., C. Chiu, S. Huang, T. Muir, Y. Zhao, B. Chait, P. Mathias, and G. Nemerow. 1997. Localization of an integrin binding motif (RGD) on adenovirus type 2 particles by cryo-electron microscopy. EMBO J. 16:1189-1198[Medline].
32. Suzuki, S., W. S. Argraves, R. Pytela, H. Arai, T. Krusius, M. D. Pierschbacher, and E. Ruoslahti. 1986. cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesion protein receptors. Proc. Natl. Acad. Sci. USA 83:8614-8618[Abstract/Free Full Text].
33. Tomko, R. P., R. Xu, and L. Philipson. 1997. HCAR and MAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc. Natl. Acad. Sci. USA 94:3352-3356[Abstract/Free Full Text].
34. Vogel, B. E., S.-J. Lee, A. Hildebrand, W. Craig, M. D. Pierschbacher, F. Wong-Staal, and E. Ruoslahti. 1993. A novel integrin specificity exemplified by binding of the alpha vbeta 5 integrin to the basic domain of the HIV tat protein and vitronectin. J. Cell Biol. 121:461-468[Abstract/Free Full Text].
35. Weinacker, A., A. Chen, M. Agrez, R. I. Cone, S. Nishimura, E. Wayner, R. Pytela, and D. Sheppard. 1994. Role of the integrin alpha vbeta 6 in cell attachment to fibronectin. J. Biol. Chem. 269:1-9[Free Full Text].
36. Wickham, T. J., P. Mathias, D. A. Cheresh, and G. R. Nemerow. 1993. Integrins alpha vbeta 3 and alpha vbeta 5 promote adenovirus internalization but not virus attachment. Cell 73:309-319[Medline].

Journal of Virology, November 1998, p. 8669-8675, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Lindert, S., Silvestry, M., Mullen, T.-M., Nemerow, G. R., Stewart, P. L. (2009). Cryo-Electron Microscopy Structure of an Adenovirus-Integrin Complex Indicates Conformational Changes in both Penton Base and Integrin. J. Virol. 83: 11491-11501 [Abstract] [Full Text]  
  • Wu, E., Trauger, S. A., Pache, L., Mullen, T.-M., Von Seggern, D. J., Siuzdak, G., Nemerow, G. R. (2004). Membrane Cofactor Protein Is a Receptor for Adenoviruses Associated with Epidemic Keratoconjunctivitis. J. Virol. 78: 3897-3905 [Abstract] [Full Text]  
  • Salone, B., Martina, Y., Piersanti, S., Cundari, E., Cherubini, G., Franqueville, L., Failla, C. M., Boulanger, P., Saggio, I. (2003). Integrin {alpha}3{beta}1 Is an Alternative Cellular Receptor for Adenovirus Serotype 5. J. Virol. 77: 13448-13454 [Abstract] [Full Text]  
  • Wu, E., Pache, L., Von Seggern, D. J., Mullen, T.-M., Mikyas, Y., Stewart, P. L., Nemerow, G. R. (2003). Flexibility of the Adenovirus Fiber Is Required for Efficient Receptor Interaction. J. Virol. 77: 7225-7235 [Abstract] [Full Text]  
  • Chiu, C. Y., Wu, E., Brown, S. L., Von Seggern, D. J., Nemerow, G. R., Stewart, P. L. (2001). Structural Analysis of a Fiber-Pseudotyped Adenovirus with Ocular Tropism Suggests Differential Modes of Cell Receptor Interactions. J. Virol. 75: 5375-5380 [Abstract] [Full Text]  
  • Miyazawa, N., Crystal, R. G., Leopold, P. L. (2001). Adenovirus Serotype 7 Retention in a Late Endosomal Compartment prior to Cytosol Escape Is Modulated by Fiber Protein. J. Virol. 75: 1387-1400 [Abstract] [Full Text]  
  • Legler, D., Wiedle, G, Ross, F., Imhof, B. (2001). Superactivation of integrin (&agr;)v(&bgr;)3 by low antagonist concentrations. J. Cell Sci. 114: 1545-1553 [Abstract]  
  • Isberg, R., Barnes, P (2001). Subversion of integrins by enteropathogenic Yersinia. J. Cell Sci. 114: 21-28 [Abstract]  
  • Russell, W. C. (2000). Update on adenovirus and its vectors. J. Gen. Virol. 81: 2573-2604 [Full Text]  
  • Arnberg, N., Kidd, A. H., Edlund, K., Olfat, F., Wadell, G. (2000). Initial Interactions of Subgenus D Adenoviruses with A549 Cellular Receptors: Sialic Acid versus alpha v Integrins. J. Virol. 74: 7691-7693 [Abstract] [Full Text]  
  • Nakano, M. Y., Boucke, K., Suomalainen, M., Stidwill, R. P., Greber, U. F. (2000). The First Step of Adenovirus Type 2 Disassembly Occurs at the Cell Surface, Independently of Endocytosis and Escape to the Cytosol. J. Virol. 74: 7085-7095 [Abstract] [Full Text]  
  • Li, E., Stupack, D. G., Brown, S. L., Klemke, R., Schlaepfer, D. D., Nemerow, G. R. (2000). Association of p130CAS with Phosphatidylinositol-3-OH Kinase Mediates Adenovirus Cell Entry. J. Biol. Chem. 275: 14729-14735 [Abstract] [Full Text]  
  • Kirby, I., Davison, E., Beavil, A. J., Soh, C. P. C., Wickham, T. J., Roelvink, P. W., Kovesdi, I., Sutton, B. J., Santis, G. (2000). Identification of Contact Residues and Definition of the CAR-Binding Site of Adenovirus Type 5 Fiber Protein. J. Virol. 74: 2804-2813 [Abstract] [Full Text]  
  • Nemerow, G. R., Stewart, P. L. (1999). Role of alpha v Integrins in Adenovirus Cell Entry and Gene Delivery. Microbiol. Mol. Biol. Rev. 63: 725-734 [Abstract] [Full Text]  
  • Chiu, C. Y., Mathias, P., Nemerow, G. R., Stewart, P. L. (1999). Structure of Adenovirus Complexed with Its Internalization Receptor, alpha vbeta 5 Integrin. J. Virol. 73: 6759-6768 [Abstract] [Full Text]  
  • Huang, S., Reddy, V., Dasgupta, N., Nemerow, G. R. (1999). A Single Amino Acid in the Adenovirus Type 37 Fiber Confers Binding to Human Conjunctival Cells. J. Virol. 73: 2798-2802 [Abstract] [Full Text]  
  • Coe, A. P. F., Askari, J. A., Kline, A. D., Robinson, M. K., Kirby, H., Stephens, P. E., Humphries, M. J. (2001). Generation of a Minimal alpha 5beta 1 Integrin-Fc Fragment. J. Biol. Chem. 276: 35854-35866 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mathias, P.
Right arrow Articles by Nemerow, G. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mathias, P.
Right arrow Articles by Nemerow, G. R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»