Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA

Prem Seth, Melissa Rosenfeld, James Higginbotham, Ronald Crystal

Research output: Contribution to journalArticle

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Abstract

Given the knowledge that replication-deficient adenoviruses can mediate the delivery of unlinked plasmid DNA into eukaryotic cells (K. Yoshimura, M. A. Rosenfeld, P. Seth, and R. G. Crystal, J. Biol. Chem. 268:2300-2303, 1993), this study focuses on the role of receptor-mediated endocytosis in this process. AdCFTR (an E1- E3- adenovirus type 5-based replication- deficient adenovirus containing the 4.5-kb human cystic fibrosis transmembrane conductance regulator cDNA) was added to Cos-7 cells together with plasmid pRSVL (containing the Rous sarcoma virus long terminal repeat promoter followed by the luciferase cDNA), and luciferase activity was quantified as a measure of the expression of the plasmid DNA. When AdCFTR was bound to Cos-7 cells at 4°C and the cells were subsequently incubated at 37°C in the presence of pRSVL, the expression of luciferase activity was increased in proportion to the amount of AdCFTR added, reaching >104-fold at 3,000 PFU per cell. AdCFTR-mediated increase in pRSVL was inhibited by addition of purified adenovirus fiber but not hexon, suggesting cell surface adenovirus receptors were involved in the cointernalization process. Cell lines with a high number of adenovirus receptors (Cos-7 and HeLa) showed significant AdCFTR-dependent pRSVL expression, while cell lines with low numbers of adenovirus receptors (NIH 3T3 and U-937) showed little. AdCFTR- mediated increase in the expression of pRSVL was prevented when AdCFTR was heat treated and exposed to antibody against adenovirus or when the cointernalization process was evaluated in the presence of chloroquine, conditions all known to prevent adenovirus-mediated disruption of endocytic vesicles. In contrast, the uptake of AdCFTR into Cos-7 cells was not affected by any of these conditions. When AdCFTR was exposed to UV light, its ability to grow in 293 cells was obviated, but AdCFTR-dependent increase in pRSVL expression was minimally reduced. Finally, empty capsids of AdCFTR were able to enhance the delivery and expression of plasmid pRSVL into Cos-7 cells, suggesting that the adenovirus genome is not required for AdCFTR-mediated plasmid cointernalization. Together, these observations suggest that the ability of a replication-deficient recombinant adenovirus to mediate the cointernalization and expression of plasmids is mediated by the receptor- mediated endocytosis pathway.

Original languageEnglish
Pages (from-to)933-940
Number of pages8
JournalJournal of Virology
Volume68
Issue number2
Publication statusPublished - 1 Feb 1994
Externally publishedYes

Fingerprint

Adenoviridae
plasmids
Plasmids
DNA
Luciferases
luciferase
receptors
cells
Endocytosis
endocytosis
Complementary DNA
Rous sarcoma virus
Cell Line
Transport Vesicles
Cystic Fibrosis Transmembrane Conductance Regulator
Terminal Repeat Sequences
Capsid
Chloroquine
Cell Surface Receptors
Eukaryotic Cells

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA. / Seth, Prem; Rosenfeld, Melissa; Higginbotham, James; Crystal, Ronald.

In: Journal of Virology, Vol. 68, No. 2, 01.02.1994, p. 933-940.

Research output: Contribution to journalArticle

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