Identification of a Conserved Receptor-Binding Site on the Fiber Proteins of CAR-Recognizing Adenoviridae

Roelvink, Peter W.; Lee, Gai Mi; Einfeld, David A.; Kovesdi, Imre; Wickham, Thomas J.

doi:10.1126/science.286.5444.1568

Cited by 367 publications

(283 citation statements)

References 22 publications

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“…As this site is located on the side of the knob monomer, these findings imply that the trimeric knob could bind to three CAR molecules independently. 18 Bewley et al, 19 by analyzing the crystal structure of the CAR-recognizing Ad12 fiber knob complexed with the D1 domain of the CAR protein, confirmed that the AB loop of the fiber plays an essential role in the interaction with the cellular receptor. Another group also confirmed, by surface plasmon resonance and Gene Therapy cell-binding competition experiments with recombinant mutated Ad fibers, that residues located in the AB and the DE loops and in ␤-strand E and F are directly or indirectly in contact with the cellular receptor.…”

Section: Introductionmentioning

confidence: 96%

“…15,16 Only recently, through sequence analysis and mutagenesis of the knob domain of the Ad5, has a conserved receptor-binding region been identified. [17][18][19][20][21] These authors demonstrated that the CARbinding site consists of residues from the AB loop, the B ␤-sheet and the DE loop, while the FG loop seems to play an indirect role in binding CAR. As this site is located on the side of the knob monomer, these findings imply that the trimeric knob could bind to three CAR molecules independently.…”

Section: Introductionmentioning

confidence: 99%

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Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

et al. 2001

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Targeting of adenovirus (Ad)-encoded therapeutic genes to specific cell types has become a major goal in gene therapy. Redirecting the specificity of infection requires the abrogation of the natural interaction between the viral fiber and its cellular receptors (CAR) and the simultaneous introduction of a new binding specificity into the viral capsid. To abrogate the natural affinity of the fiber, we have mutated residues presumed to be directly or indirectly involved in CARbinding in the knob domain of the fiber protein. These residues are located in the AB loop (Ser408) and in the DG loop (Tyr491, Ala494, Ala503). The mutations Ser408Glu,

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Section: Introductionmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 99%

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

et al. 2001

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“…5,[12][13][14] When an adenovirus particle enters a target cell, two types of receptor-mediated process are involved. First, the fiber knob of the adenovirus capsid binds to the attachment receptor on the cell surface, such as a Coxsackie B and adenovirus receptor (CAR) [15][16][17][18] and a MHC class I ␣2 domain. 19 The CAR has been reported as a 46 kDa transmembrane protein and hCAR cDNA and LacZ gene.…”

Section: Introductionmentioning

confidence: 99%

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA

et al. 2001

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“…However, there is growing evidence that the expression of CAR is frequently suppressed in various types of cells, including tumor cells and in primary tumors [4,5], resulting in resistance to adenovirus infection [6][7][8]. Nevertheless, various studies have shown that adenovectors with modified capsid proteins, such as additions of polylysine or RGD sequence to fibers, can effectively infect cells with defective CAR expression [9][10][11]. Thus, modified adenovectors with increased transduction ability or targeting capacity may be useful for various gene transfer or gene therapy studies or clinical applications.…”

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confidence: 99%

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Peng¹,

Wu²,

Davis³

et al. 2006

Analytical Biochemistry

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Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and sizeexclusion column chromatography. The purity and infectious activity of adenovectors isolated by either method were comparable. The new method yielded three to four times more adenovectors with RGD-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors, but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3 hours. Therefore, this method is rapid and efficient for production recombination adenovectors, especially those with RGD-modified fibers.Adenoviral vectors (adenovectors) have high in vivo transduction efficiencies and are easy to construct and produce. They are thus frequently used for in vitro and in vivo gene delivery and for gene therapy in clinical trials [1] . Substantial efforts have been made to characterize adenovirus-host cell interactions and to improve gene delivery by adenovectors, including minimizing vector gene expression, reducing vector related toxicity and immunogenicity, increasing their capacity to accommodate large foreign genes, and increasing their transduction efficiency.It is now known that adenovirus interacts with host cells by binding to the coxsackievirus and adenovirus receptor (CAR) [2], a cellular receptor for Ad5 and most other adenovirus serotypes. After attachment, the virion is then internalized by endocytosis through the interaction of its penton base with α v β 3 and α v β 5 integrins on the host cell surface [3]. However, there is growing evidence that the expression of CAR is frequently suppressed in various types of cells, including tumor cells and in primary tumors [4,5], resulting in resistance to adenovirus infection [6][7][8] Unfortunately, technical difficulties may be encountered in production of certain modified adenovectors. We have encountered difficulties when producing certain adenovectors by this method, especially those with RGD-modified fibers or "sticky" virus. For example, we have experienced difficulty in purifying various RGD-modified adenoviral vectors by conventional two-cycle (2x) of CsCl ultracentrifugation because the RGD-modified adenoviral vectors tend to aggregate, especially in the second round of ultracentrifugation, forming floccules without a sharp band. This results in a low yield or even failure of purification. Thus, an alternative method for efficient production of these modified adenov...

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Identification of a Conserved Receptor-Binding Site on the Fiber Proteins of CAR-Recognizing Adenoviridae

Cited by 367 publications

References 22 publications

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

Influence of adenoviral fiber mutations on viral encapsidation, infectivity and in vivo tropism

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

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