Manipulation of the cytoplasmic and transmembrane domains alters cell surface levels of the coxsackie-adenovirus receptor and changes the efficiency of adenovirus infection

W. Van't Hof, Ronald Crystal

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Expression of the coxsackie-adenovirus receptor (CAR) is a critical determinant in cellular susceptibility to infection with adenovirus-based gene transfer vectors. This study is focused on the hypothesis that manipulation of the cytoplasmic tail and transmembrane regions of CAR can be used to change cell surface levels of CAR and, consequently, to alter the efficiency of Ad-mediated gene transfer. To accomplish this, Flag-tagged ([F]) human CAR ([F]CAR), [F]tailless-CAR (lacking the cytoplasmic tail), and [F]GPI-CAR (containing a GPI lipid anchor instead of the transmembrane and cytoplasmic regions) were exogenously expressed in CHO cells. Analysis of 125I-labeled anti-Flag antibody binding to transfected cells revealed that [F]tailless-CAR and [F]GPI-CAR were expressed on the cell surface in 1.8- to 2.5-fold higher amounts than [F]CAR, while the total expression levels were similar. Infection with replication-deficient adenovirus encoding β-galactosidase (Adβgal) demonstrated 1.5- to 2-fold higher levels of transgene expression in CHO cells expressing [F]tailless-CAR or [F]GPI-CAR, respectively, compared with cells containing [F]CAR. The form of CAR expressed did not affect the transport of fluorescent Cy3-Ad particles from the cell surface to the nuclear region. These observations indicate that transduction of target cells by Ad vectors can be optimized by increasing cell surface levels of CAR through functional deletion of the tail and membrane protein domains.

Original languageEnglish
Pages (from-to)25-34
Number of pages10
JournalHuman Gene Therapy
Volume12
Issue number1
DOIs
Publication statusPublished - 1 Jan 2001
Externally publishedYes

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Coxsackie and Adenovirus Receptor-Like Membrane Protein
Adenoviridae Infections
CHO Cells
Galactosidases
Human Adenoviruses

ASJC Scopus subject areas

  • Genetics

Cite this

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title = "Manipulation of the cytoplasmic and transmembrane domains alters cell surface levels of the coxsackie-adenovirus receptor and changes the efficiency of adenovirus infection",
abstract = "Expression of the coxsackie-adenovirus receptor (CAR) is a critical determinant in cellular susceptibility to infection with adenovirus-based gene transfer vectors. This study is focused on the hypothesis that manipulation of the cytoplasmic tail and transmembrane regions of CAR can be used to change cell surface levels of CAR and, consequently, to alter the efficiency of Ad-mediated gene transfer. To accomplish this, Flag-tagged ([F]) human CAR ([F]CAR), [F]tailless-CAR (lacking the cytoplasmic tail), and [F]GPI-CAR (containing a GPI lipid anchor instead of the transmembrane and cytoplasmic regions) were exogenously expressed in CHO cells. Analysis of 125I-labeled anti-Flag antibody binding to transfected cells revealed that [F]tailless-CAR and [F]GPI-CAR were expressed on the cell surface in 1.8- to 2.5-fold higher amounts than [F]CAR, while the total expression levels were similar. Infection with replication-deficient adenovirus encoding β-galactosidase (Adβgal) demonstrated 1.5- to 2-fold higher levels of transgene expression in CHO cells expressing [F]tailless-CAR or [F]GPI-CAR, respectively, compared with cells containing [F]CAR. The form of CAR expressed did not affect the transport of fluorescent Cy3-Ad particles from the cell surface to the nuclear region. These observations indicate that transduction of target cells by Ad vectors can be optimized by increasing cell surface levels of CAR through functional deletion of the tail and membrane protein domains.",
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