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Integrin txv/35 Selectively Promotes Adenovirus Mediated Cell Membrane Permeabilization

Rockefeller University Press
Journal of Cell Biology (JCB)
Authors:
Thomas Wickham at Rubius Therapeutics
Thomas Wickham
Edward J. Filardo
Edward J. Filardo
Edward J. Filardo
  • This person is not on ResearchGate, or hasn't claimed this research yet.
David Cheresh at University of California, San Diego
David Cheresh
Glen R. Nemerow
Glen R. Nemerow
Glen R. Nemerow
  • This person is not on ResearchGate, or hasn't claimed this research yet.
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Abstract and Figures

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.
Adenovirus-induced permeabilization of HEp-2 cells adhered to cell matrix proteins or penton base. [3H]choline-labeled HEp-2 cells were adhered to laminin, vitronectin, or penton basecoated wells and then incubated with varying amounts of purified Ad2 at 4°C for 60 min. The cells were then washed and incubated at 37°C for 60 min in permeabilization buffer at pH 6.0. The amount of [3H]choline released into the buffer or remaining with the cells was measured in a scintillation counter. Nonspecific [3H]choline release (cells not exposed to Ad2) was ,,o10% of the total cellassociated cpm and was independent of the cell matrix protein used. The amount of [JH]choline corresponding to 100% was 1,954, 6,029, and 13,881 cpm for 5, 15, and 40 #g of Ad2, respectively. The total cell-associated [3H]choline was 51,000 cpm. The results represent the average of duplicate samples.
Adenovirus-induced permeabilization of HEp-2 cells adhered to cell matrix proteins or penton base. [3H]choline-labeled HEp-2 cells were adhered to laminin, vitronectin, or penton basecoated wells and then incubated with varying amounts of purified Ad2 at 4°C for 60 min. The cells were then washed and incubated at 37°C for 60 min in permeabilization buffer at pH 6.0. The amount of [3H]choline released into the buffer or remaining with the cells was measured in a scintillation counter. Nonspecific [3H]choline release (cells not exposed to Ad2) was ,,o10% of the total cellassociated cpm and was independent of the cell matrix protein used. The amount of [JH]choline corresponding to 100% was 1,954, 6,029, and 13,881 cpm for 5, 15, and 40 #g of Ad2, respectively. The total cell-associated [3H]choline was 51,000 cpm. The results represent the average of duplicate samples.
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Adhesive properties of CS-1 cells expressing txvB3 and txvB5. [3H]Thymidine labeled CS-1 cells (105) expressing c~vB3
Adhesive properties of CS-1 cells expressing txvB3 and txvB5. [3H]Thymidine labeled CS-1 cells (105) expressing c~vB3
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Susceptibility of CS-1//53 and CS-1//55 cells to adenovirus infection. CS-1//53 and CS-1//35 cells (2 x 104) were incubated
Susceptibility of CS-1//53 and CS-1//55 cells to adenovirus infection. CS-1//53 and CS-1//35 cells (2 x 104) were incubated
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Inhibition of Ad2 infection of CS-1//35 cells by function blocking mAbs to c~vB5. CS-1/B5 cells (2 × 104) were preincubated with 500/~g/ml mAbs to t~vB5 (P3G2), c~v/33 (LM609) or /31 integrins (P4C10) followed by incubation with Ad2 at an MOI of 100. Virus infection was assessed by the fluorescent focus assay.
Inhibition of Ad2 infection of CS-1//35 cells by function blocking mAbs to c~vB5. CS-1/B5 cells (2 × 104) were preincubated with 500/~g/ml mAbs to t~vB5 (P3G2), c~v/33 (LM609) or /31 integrins (P4C10) followed by incubation with Ad2 at an MOI of 100. Virus infection was assessed by the fluorescent focus assay.
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Inhibition of Ad2-induced permeabilization of CS-1//35 cells by soluble penton base and anti-c~v/$5 mAb. [3H]Choline- labeled CS-1//35 ceils were adhered to laminin-coated wefts and then incubated with 1 mg/ml soluble penton base, 200 ~g/ml mAb P3G2 (anti-o~v/~5), LM142 (a nonfunctional blocking rnAb to c~v integrins) or P4C10. The ceils were then washed and incubated with 20 #g/ml Ad2 at 4°C for 1 h and then in permeabilization buffer for 1 h at 37°C. Nonspecific release of [3H]choline was ,x,5%. The results represent the average of duplicate samples.
+1
Inhibition of Ad2-induced permeabilization of CS-1//35 cells by soluble penton base and anti-c~v/$5 mAb. [3H]Choline- labeled CS-1//35 ceils were adhered to laminin-coated wefts and then incubated with 1 mg/ml soluble penton base, 200 ~g/ml mAb P3G2 (anti-o~v/~5), LM142 (a nonfunctional blocking rnAb to c~v integrins) or P4C10. The ceils were then washed and incubated with 20 #g/ml Ad2 at 4°C for 1 h and then in permeabilization buffer for 1 h at 37°C. Nonspecific release of [3H]choline was ,x,5%. The results represent the average of duplicate samples.
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  • Feb 1992
  • METHOD ENZYMOL
This chapter discusses receptor-mediated endocytosis in semiintact cells. Many biologically important macromolecules are internalized into cells with the complex, multistep process of receptor-mediated endocytosis. Bound ligands and their receptors are first clustered into specialized coated pit regions of the cell surface. Cell surface receptors internalized via coated pits fall into two classes. One class, including, transferrin and low-density lipoprotein (LDL) receptors, are constitutively localized in coated pits and efficiently internalized at rates independent of bound ligand. In contrast, unoccupied receptors such as epidermal growth factor (EGF) and insulin receptors are internalized at low basal rates. These events not only require the addition of coat constituents but are accompanied by morphological changes in the coat, which progresses from an initially planar structure to a highly curved, invaginated pit. Finally, a membrane fission event occurs and the coat is sealed around the newly budded coated vesicle. Molecular dissection of these biochemically diverse events is facilitated by the use of cell-free assays that allow distinct stages to be assayed independently. In the chapter, the assay described was used to examine receptor-mediated endocytosis in A431 cells. These assays allow independent measurement of coated pit assembly, invagination, and coated vesicle budding.
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Integrins ctv/33 and o v/35 Contribute to Cell Attachment to Vitronectin but Differentially Distribute on the Cell Surface
Article
  • Jun 1991
We investigated the role of the integrins alpha v beta 3 and alpha v beta 5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both alpha v-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with alpha v beta 5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to alpha v beta 5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only alpha v beta 3 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while alpha v beta 5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface.
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Adenovirus-Mediated Transfer of a Recombinant alpha1Antitrypsin Gene to the Lung Epithelium in Vivo
Article
  • May 1991
The respiratory epithelium is a potential site for somatic gene therapy for the common hereditary disorders alpha 1-antitrypsin (alpha 1AT) deficiency and cystic fibrosis. A replication-deficient adenoviral vector (Ad-alpha 1AT) containing an adenovirus major late promoter and a recombinant human alpha 1AT gene was used to infect epithelial cells of the cotton rat respiratory tract in vitro and in vivo. Freshly isolated tracheobronchial epithelial cells infected with Ad-alpha 1AT contained human alpha 1AT messenger RNA transcripts and synthesized and secreted human alpha 1AT. After in vivo intratracheal administration of Ad-alpha 1AT to these rats, human alpha 1AT messenger RNA was observed in the respiratory epithelium, human alpha 1AT was synthesized and secreted by lung tissue, and human alpha 1AT was detected in the epithelial lining fluid for at least 1 week.
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