Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

Adenovirus endocytosis

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.     

Adenovirus endocytosis

Pathogen entry into cells occurs by direct penetration of the plasma membrane, clathrin-mediated endocytosis, caveolar endocytosis, pinocytosis or macropinocytosis. For a particular agent, the infectious pathways are typically restricted, reflecting a tight relationship with the host. Here, we survey the uptake process of human adenovirus (Ad) type 2 and 5 and integrate it into the cell biology of endocytosis. Ad2 and Ad5 naturally infect respiratory epithelial cells. They bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors and this triggers a variety of cell responses, including endocytosis. Ad2/Ad5 endocytosis is clathrin-mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process is induced simultaneously with viral uptake, macropinocytosis. Together, these pathways are associated with viral infection. Macropinocytosis requires integrins, F-actin, protein kinase C and small G-proteins of the Rho family, but not dynamin. Macropinocytosis per se is not required for viral uptake into epithelial cells, but it appears to be a productive entry pathway of Ad artificially targeted to the high-affinity Fcgamma receptor CD64 of hematopoietic cells lacking CAR. In epithelial and hematopoietic cells, the macropinosomal contents are released to the cytosol. This requires viral signalling from the surface and coincides with particle escape from endosomes and infection. It emerges that incoming Ad2 and Ad5 distinctly modulate the endocytic trafficking and disrupt selective cellular compartments. These features can be exploited for effective artificial targeting of Ad vectors to cell types of interest.     

Reducing the Native Tropism of Adenovirus Vectors Requires Removal of both CAR and Integrin Interactions

The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and alphav integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack alphav integrin binding, or lack both CAR and alphav integrin binding. These vectors have been used to examine the roles of CAR and alphav integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of alphav integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and alphav integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and alphav integrins can impact vector distribution in vivo. Disruption of both CAR and alphav integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.     

Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors

BACKGROUND AND AIMS: While mature enterocytes are resistant to transduction by adenovirus type 5 (Ad5) vectors, undifferentiated cells are transduced much more efficiently. Our purpose was to study enterocyte transduction in models of intestinal wound healing. METHODS: Transduction was studied ex vivo using cultures of endoscopic biopsies and in vitro utilizing Caco-2 cells in models of mucosal wound healing. Vectors carried either the LacZ or the luciferase gene. CAR (coxsackievirus and adenovirus receptor) and integrins were studied with transduction inhibition and immunofluorescent staining. RESULTS: Increased transduction efficiency was observed for a subset of enterocytes with a flattened de-differentiated phenotype present at the edge of cultured biopsies. In the in vitro systems, expanding Caco-2 cell monolayers exhibited increased transducibility that was time- and dose-dependent, reaching virtually 100% in cells along the leading edge at high viral load. Bioluminescence activity of transduced expanding monolayers was up to 3-fold greater than that of non-expanding monolayers ('fence' system, 48 h, MOI 1000, p < 0.05). Mitomycin C pre-treatment did not affect levels of transduction in expanding monolayers. At the highest viral load tested, CAR or integrin blocking prior to virus application resulted in 39.4% and 45.4% reduction in transduction levels (p < 0.05). Immunofluorescence revealed altered expression of CAR on the migrating edge of the Caco-2 cultures and the expression of CAR on the apical membrane of biopsy enterocytes. CONCLUSIONS: Increased CAR and integrin accessibility in migrating enterocytes mediates increased transduction by Ad5 vectors. This subset of enterocytes provides a target for the delivery of genes of interest for both research and gene therapy applications.     

Enhancement of anti-DIII antibodies by the C3d derivative P28 results in lower viral titers and augments protection in mice

Background

Viruses bind to specific cellular receptors in order to infect their hosts. The specific receptors a virus uses are important factors in determining host range, cellular tropism, and pathogenesis. For adenovirus, the existing model of entry requires two receptor interactions. First, the viral fiber protein binds Coxsackie and Adenovirus Receptor (CAR), its primary cellular receptor, which docks the virus to the cell surface. Next, viral penton base engages cellular integrins, coreceptors thought to be required exclusively for internalization and not contributing to binding. However, a number of studies reporting data which conflicts with this simple model have been published. These observations have led us to question the proposed two-step model for adenovirus infection.

Results

In this study we report that cells which express little to no CAR can be efficiently transduced by adenovirus. Using competition experiments between whole virus and soluble viral fiber protein or integrin blocking peptides, we show virus binding is not dependent on fiber binding to cells but rather on penton base binding cellular integrins. Further, we find that binding to low CAR expressing cells is inhibited specifically by a blocking antibody to integrin αvβ5, demonstrating that in these cells integrin αvβ5 and not CAR is required for adenovirus attachment. The binding mediated by integrin αvβ5 is extremely high affinity, in the picomolar range.

Conclusions

Our data further challenges the model of adenovirus infection in which binding to primary receptor CAR is required in order for subsequent interactions between adenovirus and integrins to initiate viral entry. In low CAR cells, binding occurs through integrin αvβ5, a receptor previously thought to be used exclusively in internalization. We show for the first time that integrin αvβ5 can be used as an alternate binding receptor.     

Reduction of natural adenovirus tropism to mouse liver by fiber-shaft exchange in combination with both CAR- and alphav integrin-binding ablation

The primary receptor, the coxsackievirus and adenovirus receptor (CAR), and the secondary receptor, αv integrins, are the tropism determinants of adenovirus (Ad) type 5. Inhibition of the interaction of both the fiber with CAR and the penton base with the αv integrin appears to be crucial to the development of targeted Ad vectors, which specifically transduce a given cell population. In this study, we developed Ad vectors with ablation of both CAR and αv integrin binding by mutating the fiber knob and the RGD motif of the penton base. We also replaced the fiber shaft domain with that derived from Ad type 35. High transduction efficiency in the mouse liver was suppressed approximately 130- to 270-fold by intravenous administration of the double-mutant Ad vectors, which mutated two domains each of the fiber knob and shaft and the RGD motif of the penton base compared with those of conventional Ad vectors (type 5). Most significantly, the triple-mutant Ad vector containing the fiber knob with ablation of CAR binding ability, the fiber shaft of Ad type 35, and the penton base with a deletion of the RGD motif mediated a >30,000-fold lower level of mouse liver transduction than the conventional Ad vectors. This triple-mutant Ad vector also mediated reduced transduction in other organs (the spleen, kidney, heart, and lung). Viral DNA analysis showed that systemically delivered triple-mutant Ad vector was primarily taken up by liver nonparenchymal cells and that most viral DNAs were easily degraded, resulting in little gene expression in the liver. These results suggest that the fiber knob, fiber shaft, and RGD motif of the penton base each plays an important role in Ad vector-mediated transduction to the mouse liver and that the triple-mutant Ad vector exhibits little tropism to any organs and appears to be a fundamental vector for targeted Ad vectors.     

β1 integrins regulate fibroblast chemotaxis through control of N-WASP stability

Chemotactic migration of fibroblasts towards growth factors, such as during development and wound healing, requires precise spatial coordination of receptor signalling. However, the mechanisms regulating this remain poorly understood. Here, we demonstrate that β1 integrins are required both for fibroblast chemotaxis towards platelet-derived growth factor (PDGF) and growth factor-induced dorsal ruffling. Mechanistically, we show that β1 integrin stabilises and spatially regulates the actin nucleating endocytic protein neuronal Wiskott–Aldrich syndrome protein (N-WASP) to facilitate PDGF receptor traffic and directed motility. Furthermore, we show that in intact cells, PDGF binding leads to rapid activation of β1 integrin within newly assembled actin-rich membrane ruffles. Active β1 in turn controls assembly of N-WASP complexes with both Cdc42 and WASP-interacting protein (WIP), the latter of which acts to stabilise the N-WASP. Both of these protein complexes are required for PDGF internalisation and fibroblast chemotaxis downstream of β1 integrins. This represents a novel mechanism by which integrins cooperate with growth factor receptors to promote localised signalling and directed cell motility.     

Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases

Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.     

Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the alphav integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on alphav integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn-expressing cells. Surprisingly, 30-50% of the endosomal contents were released into the cytosol of control and also K44A-dyn-expressing cells, and the number of fluid phase-positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection.     

Integrins mediate the inhibitory effect of focal adhesion on angiotensin II-induced p44/42 mitogen-activated protein (MAP) kinase activity in human mesangial cells.

Previously, we reported that the formation of focal adhesion accelerated by accumulation of extracellular matrices may inhibit the angiotensin II-stimulated proliferation of human mesangial cells (HMCs). The process is regulated by p44/42 MAP kinase activity through the mediation of paxillin and GTPase activating proteins. In this report, we investigated the effect of integrin molecules on the angiotensin II-induced p44/42 MAP kinase activation in non-adherent HMCs. The results demonstrated that incubation of cells with both antibody to integrin beta(1) chain (K20) and GRGDS peptide induced integrin clustering, paxillin aggregation, and marked suppression of angiotensin II-induced p44/42 MAP kinase activation. On the other hand, incubation of cells with K20 alone induced integrin clustering without paxillin aggregation and the suppressive effect on angiotensin II-stimulated p44/42 MAP kinase activity. Our results strongly suggest the pivotal role of integrins in the inhibitory effect of focal adhesion on p44/42 MAP kinase activity, the checking system against angiotensin II-induced MAP kinase overactivation.     

Head and neck cancer cells are efficiently infected by Ad5/35 hybrid virus

BACKGROUND: Clinical gene therapy trials using standard Ad5-based vectors have thus far demonstrated limited efficacy, most likely due to low expression levels of adenoviral receptors on tumor cells. We sought to analyze adenoviral receptor expression levels on primary head and neck squamous cell carcinoma (HNSCC) cells and to determine whether adenoviral retargeting to the CD46 receptor via the Ad5/35 system would increase therapeutic potential for HNSCC. METHODS: We used flow cytometric analyses to determine adenoviral receptor expression levels on nine primary HNSCC cells collected from cancer patients. Adenoviruses Ad5.LacZ and Ad5/35.LacZ were used to analyze the differences in viral transduction both in vitro and in a HNSCC tumor mouse model. RESULTS: Flow cytometric analyses demonstrated uniformly high CD46 expression in all cells studied (85-99%). In contrast, coxsackievirus and adenovirus receptor (CAR) expression was substantially lower and highly variable (1.6-62%). Alpha(v) integrin expression was between 39-98%. In situ stainings for beta-galactosidase gene expression demonstrated that Ad5/35.LacZ was clearly more effective than Ad5.LacZ in transducing primary HNSCC cells. Quantification of beta-galactosidase expression revealed up to 65 times higher transgene expression from Ad5/35.LacZ than Ad5.LacZ. In vivo, beta-galactosidase expression was detected in a substantial area after a single intratumoral injection of Ad5/35.LacZ, whereas injection with Ad5.LacZ resulted in gene expression only in a few cells. CONCLUSIONS: Our results demonstrate that the low and variable CAR expression levels limit the therapeutic efficacy of Ad5-based strategies for HNSCC. In contrast, the effective in vivo transduction capacity of Ad5/35 warrants further development of this vector for the treatment of head and neck cancer.     

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.     

Flexibility of the adenovirus fiber is required for efficient receptor interaction

The adenovirus (Ad) fiber protein mediates Ad binding to the coxsackievirus and Ad receptor (CAR) and is thus a major determinant of viral tropism. The fiber contains three domains: an N-terminal tail that anchors the fiber to the viral capsid, a central shaft region of variable length and flexibility, and a C-terminal knob domain that binds to cell receptors. Ad type 37 (Ad37), a subgroup D virus associated with severe ocular infections, is unable to use CAR efficiently to infect host cells, despite containing a CAR binding site in its fiber knob. We hypothesized that the relatively short, inflexible Ad37 fiber protein restricts interactions with CAR at the cell surface. To test this hypothesis, we analyzed the infectivity and binding of recombinant Ad particles containing modified Ad37 or Ad5 fiber proteins. Ad5 particles equipped with a truncated Ad5 fiber or with a chimeric fiber protein comprised of the Ad5 knob fused to the short, rigid Ad37 shaft domain had significantly reduced infectivity and attachment. In contrast, placing the Ad37 knob onto the long, flexible Ad5 shaft allowed CAR-dependent virus infection and cell attachment, demonstrating the importance of the shaft domain in receptor usage. Increasing fiber rigidity by substituting the predicted flexibility modules in the Ad5 shaft with the corresponding regions of the rigid Ad37 fiber dramatically reduced both virus infection and cell attachment. Cryoelectron microscopy (cryo-EM) single-particle analysis demonstrated the increased rigidity of this chimeric fiber. These studies demonstrate that both length and flexibility of the fiber shaft regulate CAR interaction and provide a molecular explanation for the use of alternative receptors by subgroup D Ad with ocular tropism. We present a molecular model for Ad-CAR interactions at the cell surface that explains the significance of fiber flexibility in cell attachment.     

Canine adenovirus type 2 attachment and internalization: coxsackievirus-adenovirus receptor, alternative receptors, and an RGD-independent pathway

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins alpha(v)beta(5) and alpha(v)beta(3), which facilitate entry. The alpha(v) integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing alpha(M)beta(2) integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing alpha(v)beta(5) and alpha(v)beta(3) integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by alpha(v)beta(5), though transduction can be CAR and alpha(v)beta(3/5) independent but is alpha(M)beta(2), MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.     

Efficient gene transfer into human CD34(+) cells by a retargeted adenovirus vector

Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.     

Genetic Retargeting of Adenovirus: Novel Strategy Employing “Deknobbing” of the Fiber

For efficient and versatile use of adenovirus (Ad) as an in vivo gene therapy vector, modulation of the viral tropism is highly desirable. In this study, a novel method to genetically alter the Ad fiber tropism is described. The knob and the last 15 shaft repeats of the fiber gene were deleted and replaced with an external trimerization motif and a new cell-binding ligand, in this case the integrin-binding motif RGD. The corresponding recombinant fiber retained the basic biological functions of the natural fiber, i.e., trimerization, nuclear import, penton formation, and ligand binding. The recombinant fiber bound to integrins but failed to react with antiknob antibody. For virus production, the recombinant fiber gene was rescued into the Ad genome at the exact position of the wild-type (WT) fiber to make use of the native regulation of fiber expression. The recombinant virus Ad5/FibR7-RGD yielded plaques on 293 cells, but the spread through the monolayer was two to three times delayed compared to WT, and the ratio of infectious to physical particles was 20 times lower. Studies on virus tropism showed that Ad5/FibR7-RGD was able to infect cells which did not express the coxsackie-adenovirus receptor (CAR), but did express integrins. Ad5/FibR7-RGD virus infectivity was unchanged in the presence of antiknob antibody, which neutralized the WT virus. Ad5/FibR7-RGD virus showed an expanded tropism, which is useful when gene transfer to cells not expressing CAR is needed. The described method should also make possible the construction of Ad genetically retargeted via ligands other than RGD.     

Fibronectin receptor modulates cyclin-dependent kinase activity.

The high affinity fibronectin receptor (FNR) is expressed by hematopoietic cells, fibroblasts, and proliferating epidermal cells. Expression of this integrin is altered by chemical and viral transformation, suggesting that FNR dysfunction may play a role in growth control. This study demonstrates that exposing FA-K562 cells to glycine-arginine-glycine-aspartate-serine (GRGDS), a peptide ligand of the FNR, specifically stimulates p34/cdc2- and cyclin A-associated kinase activities. This occurs within 2 h of peptide addition. The 110-kDa form of the retinoblastoma protein appears within 3 h of GRGDS addition, consistent with activation of a G1/S kinase. DNA staining profiles demonstrate that GRGDS induces cell cycle progression within 24 h. Increased anchorage-independent growth is subsequently observed in GRGDS-treated FA-K562 cells. The control peptide, GRGES, which cannot bind the FNR, has none of these effects. This demonstrates that an extracellular integrin ligand can regulate cell proliferation. Furthermore, these results suggest that integrins link the extracellular environment and intracellular growth regulators.     

Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes

Adenovirus (Ad) fiber proteins are responsible for the initial attachment of the virion to the cell membrane. Most Ad vectors currently in use are based on the Ad type 5 (Ad5), which belong to subgroup C, and use the coxsackievirus and adenovirus receptors (CAR) as the initial receptor. Ad35, which belongs to subgroup B, recognizes unknown receptor(s) other than CAR. In this study, the feasibility of the Ad vector containing Ad5/35 chimeric fiber protein was examined in a wide variety of cell types, such as CAR-positive or -negative human tumor cells, rodent cells, and blood cells (a total of 20 cell types), and in mice in vivo. Transduction data suggested that the Ad vectors containing the Ad5/35 chimeric fiber protein exhibited altered and expanded tropism when compared with the Ad5-based vector. The chimeric vector also allows the packaging of larger foreign DNAs than the conventional Ad5-based vector, which can package approximately 8.1-8.2 kb of foreign DNA. The chimeric vector containing approximately 8.8 kb of foreign DNA was generated without affecting the viral growth rate and titer. These results suggested that inclusion of the Ad35 fiber protein into the Ad5-based vector could lead to an improved efficiency in gene therapy and in gene transfer experiments, especially for the cells lacking in sufficient CAR expression.