Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus infection and ad-mediated gene transfer.
CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.
Gaden
Florence
F
Laboratoire de Virologie et Pathogénèse Virale, Faculté de Médecine RTH Laennec, Lyon, France.
Franqueville
Laure
L
Hong
Saw See
SS
Legrand
Valérie
V
Figarella
Catherine
C
Boulanger
Pierre
P
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000257:Adenoviridae Infections / Q000235:genetics; D001980:Bronchi / Q000166:cytology / Q000821:virology; D002236:Carbohydrate Conformation; D002448:Cell Adhesion / Q000235:genetics; D002478:Cells, Cultured; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D003550:Cystic Fibrosis / Q000235:genetics / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004705:Endocytosis; D018014:Gene Transfer Techniques; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D009842:Oligopeptides / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics / Q000378:metabolism; D012795:Sialoglycoproteins / Q000737:chemistry / Q000378:metabolism; D014132:Trachea / Q000473:pathology* / Q000503:physiopathology / Q000821:virology; D014771:Virion / Q000378:metabolism
C505032:CFTR protein, human; C568771:CLMP protein, human; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D019812:Heparan Sulfate Proteoglycans; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D011991:Receptors, Virus; D012795:Sialoglycoproteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C047981:arginyl-glycyl-aspartic acid