Retroviral display of urokinase-binding domain fused to amphotropic envelope protein.
Tumors frequently express urokinase (uPA) receptor (uPAR). To investigate whether uPAR can efficiently target cancerous cells using amphotropic retroviral vectors, we generated a retrovirus displaying the amino-terminal fragment (ATF) of uPA as an N-terminal extension of viral envelope protein. We also made use of a "two-step strategy" by inserting a uPA cleavage site between the ATF moiety and the envelope. We measured the ability of ATF-bearing chimeric envelopes to infect huPAR-overexpressing Madin-Darby canine kidney (MDCK) and control MDCK II cells. The ATF-viruses infected both MDCK cell lines with an equivalent efficiency, suggesting that the chimeric viruses were not sequestered by uPAR and infect cells preferentially via the Pit-2 receptor. The addition of a uPA cleavage site increased the infection level of huPAR-MDCK cells by 2-fold when uPA was present in the infection medium. Surprisingly, ATF-env viruses infected huPAR-MDCK cells 5.5-fold more efficiently in the presence of exogenous uPA. This stimulatory effect of uPA on infection of huPAR-MDCK cells by the ATF-env virus was completely abolished by methyl-beta-cyclodextrin, suggesting that this effect involves the caveolar endocytosis pathway.
Boucquey
Antoine
A
EMI 00-20, INSERM and Université Paris XI, Bat Grégory Pincus, 80 rue du Général Leclerc, 94276 Le Kremlin Bicêtre Cedex, France.
Vilhardt
Frederik
F
Mitrovic
Tatjana
T
Franco
Dominique
D
Weber
Anne
A
Horellou
Philippe
P
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D012190:Retroviridae / Q000235:genetics*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*
D017931:DNA Primers; D014759:Viral Envelope Proteins; D014568:Urokinase-Type Plasminogen Activator