Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization

Human adenovirus type 2 (Ad2) enters host cells by receptor-mediated endocytosis, an event mediated by the virus penton base binding to cell surface integrins alpha v beta 3 and alpha v beta 5. While both alpha v integrins promote virus internalization, alpha v beta 5 is involved in the subsequent event of membrane permeabilization. Cells transfected with the beta 5 or beta 3 subunit, expressing either alpha v beta 5 and alpha v beta 3, respectively, were capable of supporting Ad2 infection to varying degrees. In this case, cells expressing alpha v beta 5 were significantly more susceptible to Ad2-induced membrane permeabilization, as well as to Ad2 infection, than cells expressing alpha v beta 3. Adenovirus-mediated gene delivery was also more efficient in cells expressing alpha v beta 5. These results suggest that the interaction of alpha v beta 5 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virus- mediated membrane permeabilization and suggest a novel biological role for integrin alpha v beta 5 in the infectious pathway of a human adenovirus.

tion, as well as to Ad2 infection, than cells expressing avfl3. Adenovirus-mediated gene delivery was also more efficient in cells expressing txvfl5. These results suggest that the interaction of oLv/35 with Ad2 penton base facilitates the subsequent step of virus penetration into the cell. These studies provide evidence for the involvement of a cellular receptor in virusmediated membrane permeabilization and suggest a novel biological role for integrin avfl5 in the infectious pathway of a human adenovirus.

crucial step in virus infection of host cells is pene-
tration/permeabilization of the cell plasma membrane, a past-internalization event required for delivery of the viral genome into the cytoplasm. Although a substantial amount of knowledge exists on cell entry by enveloped viruses, the mechanism(s) by which nonenveloped viruses penetrate cells is not well understood. Adenovirus, a nonenveloped DNA virus that is a major cause of respiratory and gastrointestinal infections of children (3,14), has proved useful for studying cell entry by nonenveloped viruses.
Of the over 40 different serotypes of human adenovirus, the majority of cell interaction studies have been performed with serotype 2 (human adenovirus type 2; Ad2) t. Initial attachment of Ad2 to host cells is mediated by the fiber protein (13,17), an elongated 62-kD protein that is present on each of the 12 vertices of the virion capsid (28). The fiber receptor, which is broadly distributed on a variety of cells, has not yet been identified. After Ad2 attachment to the fiber receptor, virus particles are rapidly internalized into clathrin-coated vesicles by receptor-mediated endocytosis (4,33). The fiber protein is dissociated from the virion particle early in the entry pathway (12). Ad2 internalization is mediated by either of two secondary host cell receptors, integrins olv/33 and o~v/35 (38). Ad2 binding to av integrins is mediated by five Arg-Gly-Asp (RGD) sequences present in a separate virus coat protein known as the penton base (1,38). Mutations introduced into the penton base RGD sequence abolish interaction with av integrins and also inhibit efficient virus infection (1). Thus, Ad2 cell entry requires sequential steps involving virus attachment to a primary cell receptor via the fiber protein, followed by internalization mediated by penton base binding to av integrins.
Following virus internalization, Ad2 causes disruption of cell endosomes by a pH-dependent mechanism (2,26) which is still poorly understood. Ad-mediated endosome disruption requires a low pH environment since agents such as monensin that inhibit endosome acidification also block Ad penetration but not virus internalization (12). The ability of adenovirus to disrupt cell endosomes efficiently has facilitated the use of this virus as a vector for gene therapy (20).
Ad2 also induces cell membrane permeabilization at pH 6.0 (2,23), an activity associated with virus disruption of the cell endosome (penetration). Ad2-mediated membrane permeabilization has been demonstrated by the release of fluorescent dyes from liposomes (2) or by release of 51chromium or pH]choline from human epithelial cells (23). A number of earlier studies indicated that Ad2 penton base has an important role in membrane permeabilization. Polyclonal antibodies directed against the Ad2 penton base but not fiber or hexon protein block Ad2-mediated membrane permeabilization, as measured by the release of nchromium (26) or by enhanced susceptibility to Pseudomonas toxin (22). The penton base also has a propensity to bind to nonionic detergents at low pH compared to other adenovirus coat proteins (25), suggesting that membrane-reactive hydrophobic regions in the penton base may become exposed upon genera-tion of the low pH in the endosome. The penton base is released from Ad2 virions shortly after virus internalization (12), further suggesting the penton base is involved in membrane interactions within the cell endosome.
In these studies, we established cell lines expressing either t~v/~3 or c~v/35 to analyze the role of each of these receptors in Ad2 penton base binding, internalization, and cell membrane permeabilization. The results demonstrate that although integrins av/33 and otv/35 both mediate Ad2 penton base binding and internalization, integrin o~vfl5 preferentially promotes virus-induced cell membrane permeabilization and gene delivery as well as adenovirus infection.

Cell Culture and Generation of Cell Lines
HEp-2, HeLa, and H2981 cell lines were obtained from American Type Culture Collection (Rockville, MD). The CS-1 hamster melanoma cell line (9), a generous gift of Dr. Carolyn Damsky (University of California at San Francisco, San Francisco, CA), was propagated in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 50 ttg/mi gentamicin. CS-I cells do not adhere to vitronectin due to their failure to express otv integrins (32). These cells express neither av/~3 nor av/35 beterodimers on their surface yet maintain an intracellular pool of the otv intngrin subunit. To generate CS-1 subllnes expressing either av/~3 or c~v/~5 integrins on their surface, cDNAs encoding full-length human 153 (10) or/~5 (19) subunit proteins were subcloned into the pcDNA-1/NEO expression vector (Invitrogen, La Jolla, CA) and introduced into CS-I cells via iipofectin-mediated transfection (GIBCO BRL, Gaithersburg, MD). Stably transformed cells were selected by growth in 500/~g/mi Geneticin (Sigma Chemical Co., St. Louis, MO) and enriched from the neomycin-resistant population for the expression of either c~v/33 or t~v~5 by selectively propagating the adherent cell population and discarding the unattached cells during passage of the cell line.

Virus, Antibodies, and Proteins
Ad2 was propagated in HeLa or H2981 cells and was purified and stored as previously described (8). Briefly, Ad2 was banded on cesium chloride gradients and then dialyzed against 10 mM phosphate-buffered saline, pH 8.0 containing 10% glycerol, and 1 mM MgCI2. Purified virus was stored at -70°C and dialyzed into the appropriate buffer just prior to use. Ad2 was labeled with [3I-l]thymidine as previously described (30) or with NHS-SS-biotin (Pierce Chemical Co., Rockford, IL) as recommended by the manufacturer. Recombinant adenovirus type 5 containing the gene encoding /~-glucuronidase (AdS-Glu) was kindly provided by Dr. Imi Kovesdi (Gen-Vec, Rockville, MD). AdS-glu was grown in 293 cells and purified by banding on cesium chloride gradients.
Recombinant penton base was produced in TnSB insect cells using baculovirus as previously described (38). The hexon protein, the major capsid protein of Ad2, was purified by FPLC from Ad2-infected cells using a Resource Q ion exchange column (Pharmacia Fine Chemicals, Piscataway, N J) (36). Polyclonal antibodies directed against the Ad2 fiber and penton base were produced in rabbits (38). The synthetic hexapeptides GRGDSP and GRGESP were obtained from Telios Pharmaceuticals, Inc. (San Diego, CA).

Cell Adhesion and Infection Assays
For cell adhesion assays, cells were labeled overnight with 0.5/zCi/ml of [3H]thymidine and then removed from monolayer cultures using 10 mM EDTA and resnspended in adhesion buffer (DME supplemented with 2 mM MgCI2, 1% BSA, and 20 mM Hepes). Labeled cells (1 × 10~/well) were added to individual wells of48-well nontreated cluster plates (Costar Corp., Cambridge, MA) which had been pre-coated overnight with 0.5 rng/mi pen-ton base or 3 mg/mi vitronectin and then blocked with 5 % BSA in PBS, pH 7.4 for 1 h. Cells were preincubated with 100 pg/rrd of mAbs or 150 ~tg/mi of synthetic peptides in adhesion buffer for 1 h at 4°C, and then allowed to attach to wells at 37°C for 30-60 rain. Unattached cells were removed by rapid washing with PBS, and the amount of cell-associated radioactivity remaining in each well was determined by addition of detergent and counting the cell lysates. The percentage of attached cells was calculated from the total cell cpm added to each well.
Ad2 virus infection was quantituted using the fluorescent focus assay (31). Briefly infected cell monolayers were fixed with 1% formaldehyde for 15 rain and then permeabilized with a 0.2% Triton X-100 in PBS. After washing with PBS, the cells were preblocked with 0.2% gelatin and incubated with a 1:500 dilution of polyclonai antibodies to the Ad2 fiber or penton base in PBS containing 0.2% gelatin. Cells were then washed and incubated in PBS/gelatin containing 1:500 dilution of rbodamine-labeled polyclonal goat anti-rabbit antibody (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) for 2-12 h. Infection was expressed in fluorescem focus units (FFU) which represent the number of fluorescent cells counted with a fluorescent microscope. To analyze the effect of soluble mAbs on Ad infection, cells in suspension were incubated with these agents for 1 h at 4°C followed by the addition of the appropriate amount of adenovirus at 4°C. After 1 h, the cells were then warmed to 37°C for 30 min, trypsinized for 15 min, and then plated on poly-D-lysine-coated wells. CS-I were infected in suspension with varying amounts of Ad2 for 1 h at 370C, trypsinized for 15 rain, and then washed and plated onto poly-D-lysine-coated chamber slides. For all experiments, plated cells were incubated in DME (plus 10 mM Hepes, 10% FBS) at 37°C for 2 d prior to determining the number of FFU/well.

Virus and Penton Base Binding and Internalization Assays
Binding of [3H]thymidine-labeled Ad2 or [35S]penton base to CS-1//~3 and CS-1//~5 cells was carried out as previously described (38). Nonspecific binding was determined in the presence of 50-fold excess unlabeled Ad2 or penton base.
Ad2 internalization into CS-1//33 and CS-1//~5 cells was performed with biotinylated Ad2 using a capture ELISA as previously described by Smythe et al. (27). Briefly, 60 #g of biotinylated Ad2 was added to 1 x 107 CS-1//33 or CS-1//~5 cells for 1 b at 4°C in adhesion buffer. The unbound virus was removed by washing, and cell samples of 1 x 106 cells each were warmed to 37°C for varying lengths of time. Uninternalized virus particles remaining on the cell surface were then "quenched" by the addition of 100 ~tg/mi of soluble avidin (Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 60 rain at 4°C in Hepos-buffered saline containing 10 mM EDTA. Internalized Ad2 was then released by solub'flizing the cells with 1% NP-40 in PBS/0.2 % of BSA. Cell lysates containing biotin-Ad2 were then added to ELISA wells which had been precoated with 1/~g/well of polyclonal anti-penton base IgG. Biotin-Ad2 was then detected by addition of strepavidin-alkaline pbosphatase (Boehringer-Mannheim Biocbemicals) diluted 1:1,000 in PBS/0.5% nonfat dry milk (Blotto) followed by chromogenic substrate. The total amount of Ad2-biofin bound to the cells was determined at 4°C in the absence of soluble avidin. Nonspecific binding (<I0 % of total) was determined by analyzing cells incubated in the absence of biotin-Ad2.
To analyze soluble penton base internalization, radiolabeled penton base was added to cells at 4°C and then immediately warmed to 37°C. At varying times, samples were taken and diluted 10-fold in ice-cold HBSE, (Hepesbuffered saline containing 10 mM EDTA) washed twice in this buffer, and then resuspended in a small volume of HBSE containing 2 mg/ml subtilisin (Sigma Chemical Co.) and incubated at 37°C for 15 rain. Finally, the cells were washed in ice-cold adhesion buffer, and the remaining proteaseresistant Ad2-associated cpm were measured. To determine the total cell associated penton base, the samples were washed three times in HBS containing 1 mM CaC12 and 1 mM MgCI2. Subtilisin-treated cells remained >95% viable.

Ad-induced Cell Permeabilization Assay
Cell permeability was assayed by [3H]choline release as previously described (23) with minor modifications. Experiments using CS-1 cells were performed in suspension, while permeability studies with other cell lines were performed in monolayer cultures. The appropriate number of cells in adhesion buffer containing 1-5 mCi/ml [3Hlcholine (Amersham Corp.) were incubated for 1 h at 37°C. Cells (1 × 105 cells/sample) were then washed three times with ice-cold virus binding buffer 5 mM (Hepesbuffered saline, pH 7.0 containing 0.2% BSA, 1 mM CaC12, 1 mM MgC12, and 50 mM NaN3 to prevent virus internalization during warming) (29) and incubated for 1 h at 4°C with the appropriate virus concentration (0-50 #g/ml for dose response experiments and 0-10 #g/ml for pH and kinetic experiments). After virus binding, the cell samples were washed once with saline and then incubated for 1 h at 37°C with 200 #1 permeability buffer 50 mM MES (Sigma Chemical Co.)-buffered saline, pH 6.0 containing 0.2% BSA, 1 mM CaC12, 1 mM MgCI2, and 50 mM NAN3). For pH studies, a combination of 25 mM MES and 25 mM Hepes was used to adjust the permeability buffer to the desired pH. After incubation in permeability buffer, the cell samples were incubated with or without ndenovirus and then gently centrifuged. The percent [3H]choline release was determined by measuring the counts released into the permeability buffer and the counts remaining in the cell pellet. For experiments using adherent cells, 4g-well untreated polystyrene plates were precoated with 10 mg/ml of laminin, vitronectin, or penton base in a volume of 0.2 ml. Wells were then blocked with 5% BSA and then 105 HEp-2 or CS-1 cells in adhesion buffer containing 1-5 mCi/ml [3H]choline were added to each well. Cells became adherent within 1 h, after which they were washed three times with virus-binding buffer and then incubated with adenovirus for 1 h at 4°C. Cells were then washed once with saline and incubated with permeability buffer for 1 h at 370C.
[3H]choline release was determined by measuring the radioactivity in the buffer and in the adherent cells which were solubilized in 0.2 ml of 1% SDS. For blocking experiments, antibodies or other soluble proteins were incubated with adherent cells for 1 h prior to virus binding and in the subsequent virus binding and low pH incubations.

Adenovirus-mediated Gene Delivery in CS-1 Cells
CS-1 cells, 2 x 105 in 50/~1, were infected with AdS-Glu at multiplicities of infection (MOI) of 0.1, 1.0, and 10.0 at time zero. Infected cells were incubated for 5 h in DME media and subsequently harvested and washed three times in Hepes-buffered saline. To measure expression of/3-glucuronidase, the cells were lysed by the addition of 60 #i of Reporter lysis buffer (Promega Biotec, Madison, WI) for 15 rain at 4°C. Following high-speed centrifugation, the cell lysates were analyzed for/3-glucuronidase activity using a chemiluminescence reporter assay performed according to the manufacturer's specifications (Tropix, Bedford, MA). The amount of chemilurninescence, expressed as relative light units (RLU), was measured in a LUMAT luminometer (Berthold Systems, Inc., Pittsburgh, PA).

Ad2-induced Membrane Permeabilization Involves Penton Base Interaction with ~v Integrins
A major event following adenovirus internalization into cell endosomes is penetration of the cell membrane allowing the virus nucleocapsid to enter the cytoplasm. The ability of adenovirus to permeabilize cell membranes at low pH has been used to study this event. Although our previous studies clearly showed that integrins otv/33 and otv/~5 were involved in Ad2 internalization, they did not examine whether either of these receptors also mediated virus interaction with the cell membrane. We reasoned that if integrins otv#3 and otv/~5 were involved in virus-induced membrane permeabilization, and then cells adhered to immobilized vitronectin or penton base protein should be resistant to Ad2-mediated membrane permeabilization since their otv integrins would be redistributed to the basolateral surface and thus unavailable to the virus. We have previously used this approach to demonstrate the role of otv integrins in Ad2 internalization (38). In the current studies, however, cell permeabilization studies were performed in the presence of 50 mM NaN3 to prevent virus internalization, thus restricting virus interactions to the cell plasma membrane. As shown in Fig. 1, human epithelial Hep-2 cells adhered to immobilized vitronectin or penton base were significantly less susceptible to Ad2-induced membrane permeabilization at pH 6.0 compared to cells adhered to a control matrix protein, laminin which binds to non-otv integrins. In parallel experiments, we found that cells adhered to vitronectin or penton base showed a decrease in binding of 125I-labeled mAb LM142 (634, 477 cpm, respectively) compared to cells adhered to laminin (1,007 cpm). These studies indicate that ctv integrins were redistributed to the basolateral surface following adhesion to vitronectin and penton base. These studies strongly suggested that penton base binding to otv integrin promotes Ad2-induced cell permeabilization. To examine this possibility further, we performed inhibition studies using soluble adenovirus capsid proteins or cell matrix molecules (Fig. 2). Preincubation of HEp-2 cells with soluble penton base significantly inhibited Ad2 induced membrane permeabilization, while control proteins including the Ad2 hexon protein or the cell matrix protein had no effect on Ad2 membrane permeabilization. Interestingly, penton base alone did not induce [3H]choline release from HEp-2 cells. These results suggested that penton base binding to otv integrins was required but not sufficient for Ad2-induced cell membrane permeabilization.

Adhesive Properties of CS-1 Cells Expressing o~v[33 or ~vf35
To systematically investigate the role of integrins otv/~3 and otv/~5 in the early events in Ad2 infection, a CS-1 cell model was used. CS-1 cells lack otv integrins due to the absence of the/33 or/35 integrin subunit (32). Therefore, we transfected these cells with cDNAs encoding the/33 or/35 subunit. Each of these stably transfected cell lines were capable of adhering to immobilized vitronectin or penton base (Fig. 3). Adhesion to penton base or vitronectin was blocked by the appropriate Figure 2. Inhibition of Ad2induced membrane permeabilization by soluble penton base.
[3H]choline-labeled HEp-2 cells were adhered to laminin-coated wells and then incubated at 40C for 60 rain with 1 mg/ml of purified Ad2 bexon, penton base, or collagen followed by incubation for 1 h at4°C in the absence (left) or presence of 20 ttg/ml purified Ad2 for an additional 60 min at 40C (r/ght). The cells were then incubated for 60 min at 37°C in permeabilization buffer in the presence of the indicated proteins at 1 mg/ml at pH 6.0 and assayed for [3H]choline release. The results represent the average of duplicate samples. monoclonal antibody to otvB3 (LM609) or otvB5 (P3G2) but not by a control antibody to/~1 integrins (P4C10) (Fig. 3). The parental cell line, CS-1, failed to adhere to either penton base (not shown) or vitronectin (32).

Expression of ~v/35 in CS-1 Cells Promotes Ad2-induced Membrane Perraeabilization
Since CS-1 cells expressing o~v/~3 and otvB5 were both capable of supporting Ad2 binding and internalization, we next asked whether these integrins were capable of supporting subsequent steps in infection including membrane permeabilization. As shown in Fig. 8 (Fig. 8 B) and had a pH optimum of "o6.0 (Fig. 8 C). Ad2-mediated membrane permeabilization

Integrin ave5 Promotes Adenovirus-mediated Gene Delivery
To further examine whether specific otv integrins are capable of enhancing adenovirus-penetration into the cell, we analyzed adenovirus-mediated gene delivery into CS-1 cells using a recombinant adenovirus (AdS-GLU) encoding the enzyme/3-glucuronidase. The ability of adenovirus to enhance delivery of reporter genes into cells is facilitated by virus-mediated endosome disruption (7). Delivery of the /3-glucuronidase gene and subsequent expression of the enzyme activity was measured at early times (5 h) after virus infection in the absence of virus replication. As shown in Fig. 10, av/35 expressing CS-1 cells contained 5-10-fold more fl-glucuronidase activity following infection with Ad5-GLU than cells expressing c~v/33 or the parental CS-1 cell line lacking either c~v/33 or c¢v/35. These studies, therefore, provide further evidence for the selective role of c~v/35 in promoting adenovirus penetration into cells.

Discussion
A unique feature of human adenovirus is its ability to disrupt cell endosomes efficiently thus enabling the virus to rapidly enter the cytoplasm and be translocated to the cell nucleus (4,12,33). This attribute has facilitated the use of adenovirus as a vector for gene therapy (20,21) since this virus also promotes the entry of foreign DNA into the cytoplasm before it can be degraded in lysosomes (7,35), thus overcoming one of the major impediments to gene delivery. Although the precise mechanism by which adenovirus penetrates the cell endosome has yet to be elucidated, virus-induced enhancement of cell membrane permeability has been detected by the release of small molecules such as [3H]choline phosphate from intact cells (23,26). Enhancement of cell membrane permeability is thought to be indicative of events leading to virus penetration of the cell endosome since it occurs with increasing time of incubation at 37°C and also requires mildly acidic conditions (23).
A number of previous studies indicated that the penton base protein is involved in early events leading to disruption of the cell endosome. However these earlier studies did not clearly distinguish between penton base-mediated virus internalization from virus penetration into the cytoplasm. Our recent studies demonstrated that the penton base mediates Ad2 internalization via interaction with integrins av/33 and ctv/35. In the studies reported here, while both av integrins promote Ad2 internalization, penton base binding to otv/35 was also shown to be involved in subsequent steps in virus infection, including cell membrane permeabilization. Redistribution of txv integrins to the basolateral surface of cultured cells inhibited Ad2-induced membrane permeabilization (Fig. 1). Membrane permeabilization was also blocked by preincubation of cells with soluble penton base (Fig. 2) im-plicating a role for otv integrin interaction with penton base in cell membrane permeabilization.
In previous studies with M21-L4 cells expressing both tavB3 and otv/35, function-blocking monoclonal antibodies to both oe¢/33 and otv/35 were required to block Ad2 internalization. However, these cells express ~20-fold more av/33 than ct~,~5, thus, it was not possible to assess the precise role of each integrin in later steps of virus infection. To analyze the individual roles of otv/33 and otv/35 in Ad2 infection, we developed a cell model comprised of CS-1 cells expressing either etv/33 or otv~5. It was of interest that CS-1//35 cells were significantly more susceptible to Ad2 infection than CS-1//33 cells, although both cell types supported similar levels of Ad2 and penton base binding and internalization (Fig. 6, 7). CS-1//35 cells also showed enhanced susceptibility to Ad2induced membrane permeabilization compared to CS-1//33 or CS-1 cells which were relatively resistant to Ad2 (Fig. 8).
The enhanced susceptibility of CS-1//35 cells to Ad2-induced membrane permeabilization was due to expression of tav/35 since it could be abrogated by function blocking antibodies to w¢/35 or by soluble penton base (Fig. 9). We have also observed similar differences in susceptibility to Ad2-induced membrane permeabilization in a number of other human cell lines. For example, M21-L4 human melanoma cells, which have relatively low levels of otv/35, showed a low level of Ad2induced membrane permeabilization while HEp-2 cells, which have significantly higher levels of av/35, were highly susceptible to [3H]choline release.
The enhanced susceptibility of CS-1//35 cells to Ad2induced cell membrane permeabilization also correlated with the enhanced susceptibility of these cells to virus infection (Fig. 4), as well as adenovirus-mediated gene delivery (Fig. 10). CS-1//35 cells were ,x,5-10-fold more susceptible to virus infection and supported higher levels of gene delivery compared to CS-1//33 cells, and infection could be abrogated by preincubation with the P3G2 mAb (anti-o~v/35) or soluble penton base. These results demonstrate that penton base binding to ave5 facilitates efficient virus penetration into the cell cytoplasm, an event required for efficient virus infection. However, these studies do not rule out the possibility that ctv/35 mediates other cellular events which do not directly impact virus penetration but which, nonetheless, promote infection.
We do not yet know the basis for the selective role of otv/35 for Ad2 infection. However, our preliminary studies show that penton base binding to otv/33 and c~v/35 is qualitatively different since binding to av/35 occurs at both low and neutral pH while otv/33 binding is significantly reduced at low pH (not shown). Moreover, av/~3 but not ctvfl5 could be eluted from a penton base affinity column with RGD peptides, suggesting that o~v/35 may bind to penton base in a non-RGD-dependent manner. Previous studies have shown that a non-RGD-containing basic region in HIV-Tat protein preferentially binds to av/35 (34). Whether a similar region is also involved in penton base to otv/35 binding has yet to be determined. Of particular relevance for the current studies, our preliminary studies show that penton base is capable of binding to CS-1 cells expressing o~v/35 but not otv/33 at pH 5.5, suggesting that the low pH environment of the endosome favors an interaction of ctv/35 with the Ad2 penton base. The preferential interaction of tav/35 with Ad2 penton base appears to have physiological significance since human bron-chial epithelial cells have been reported to express high levels of otv/35 and/or otv/36 while they express low or undetectable levels of txv/33 (16).
Although penton base binding to otv~/5 was shown to be required for Ad2-mediated membrane permeabilization, the penton base protein alone did not induce membrane permeabilization (Fig. 2). A possible explanation for the inability of penton base to permeabilize ceils is that other adenovirus capsid proteins (28) mediate the penetration event. However, it is also possible that penton base has an indirect role in endosome disruption through a cell-signaling pathway. Previous studies have shown that Ad2-induced cell membrane permeabilization involves ATPase activity (24), a multisubunit enzyme involved in endosome acidification (11). Cell surface integrins, including otv/~3, are also known to stimulate a rise in intracellular pH and an influx of calcium (15). Whether penton base binding to otv integrins has a role in activating this ATPase signaling pathway remains to be determined.
These studies demonstrate the participation of a specific cellular receptor integrin otv/35 in virus-host cell interactions occurring after internalization and, therefore, provide further insight into how a nonenveloped virus penetrates the cell membrane. These findings also have important implications for the future use of adenovirus to target and deliver foreign genes into host cells.