Involvement of glycosaminoglycans in vesicular stomatitis virus G glycoprotein pseudotyped lentiviral vector-mediated gene transfer into airway epithelial cells.
BACKGROUND
The involvement of surface molecules in HIV-1-derived lentivirus (LV)-mediated transduction of airway epithelial cells has not been studied so far. The present study aimed to evaluate the role of glycosaminoglycans (GAGs) in gene transfer mediated by a third generation vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped LV vector in an in vitro model of polarized airway epithelial cells.


METHODS
Human bronchial (16HBE-S1) and tracheal (CFT1-C2) epithelial cells were grown either on plastic or on filters and transduced with the LV vector polypurine tract (PPT)-green fluoresecent protein (GFP). Zonula Occludens (ZO)-1, a marker of tight junction, and GAG localization were assessed by cytofluorimetry and confocal microscopy. Soluble GAGs and removal of cell surface GAGs were used to affect LV-mediated transduction.


RESULTS
Extensive optimization of experimental parameters (presence of polybrene during the infection, the incubation time in the presence of LV particles, period of time intercurring between infection and gene expression analysis) was carried out in plastic-adherent cells. Polybrene resulted to be cytotoxic and was not further used. In CFT1-C2 polarized cells, EGTA treatment determined a 20% decrease in transepithelial resistance, a diminished ZO-1 localization at the tight junction location and a 31% increase in GFP positive cells. Heparane sulfate was distributed evenly on the cell surface. Heparin and soluble chondroitin sulfate A and B inhibited LV-mediated transduction in a dose-dependent fashion. These results were confirmed upon enzymatic removal of GAGs from the cell surface.


CONCLUSIONS
Taken together, these results show that GAGs are involved in VSV-G LV transduction of airway epithelial cells.
Copreni
Elena
E
Institute for Experimental Treatment of Cystic Fibrosis, HS Raffaele, Milan, Italy.
Castellani
Stefano
S
Palmieri
Lucia
L
Penzo
Marianna
M
Conese
Massimo
M
D000818:Animals; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006025:Glycosaminoglycans / Q000032:analysis / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000235:genetics*
C028391:G protein, vesicular stomatitis virus; D006025:Glycosaminoglycans; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins