Mechanism of enhancement of DNA expression consequent to cointernalization of a replication-deficient adenovirus and unmodified plasmid DNA. Academic Article uri icon

Overview

MeSH

  • Animals
  • Biological Transport, Active
  • Capsid
  • Cells, Cultured
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • DNA, Recombinant
  • Genome, Viral
  • Humans
  • Luciferases
  • Membrane Proteins
  • Receptors, Virus
  • Recombinant Fusion Proteins
  • Ultraviolet Rays
  • Virus Replication

MeSH Major

  • Adenoviruses, Human
  • DNA, Viral
  • Endocytosis
  • Gene Transfer Techniques
  • Plasmids

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 degrees C and the cells were subsequently incubated at 37 degrees C in the presence of pRSVL, the expression of luciferase activity was increased in proportion to the amount of AdCFTR added, reaching > 10(4)-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 recombinantly adenovirus to mediate the cointernalization and expression of plasmids is mediated by the receptor-mediated endocytosis pathway.

publication date

  • February 1994

has subject area

  • Adenoviruses, Human
  • Animals
  • Biological Transport, Active
  • Capsid
  • Cells, Cultured
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • DNA, Recombinant
  • DNA, Viral
  • Endocytosis
  • Gene Transfer Techniques
  • Genome, Viral
  • Humans
  • Luciferases
  • Membrane Proteins
  • Plasmids
  • Receptors, Virus
  • Recombinant Fusion Proteins
  • Ultraviolet Rays
  • Virus Replication

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC236531

PubMed ID

  • 7507187

Additional Document Info

start page

  • 933

end page

  • 940

volume

  • 68

number

  • 2