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. Author manuscript; available in PMC: 2010 Feb 15.
Published in final edited form as: Cancer Res. 2009 Feb 10;69(4):1459–1468. doi: 10.1158/0008-5472.CAN-08-2628

Figure 1. Rescue and in vitro characterization of recombinant oncolytic measles viruses fully retargeted against human and murine uPAR.

Figure 1

(A) Schematic representation of the recombinant retargeted measles virus genome. The amino terminal fragment (ATF) of human or murine uPA, flanked by the SfiI/NotI restriction sites was displayed as a C-terminal extension of HAALS, a measles virus H glycoprotein with 4 residue mutations (AALS: Y481A, R533A, S548L, F549S) that ablate its ability to bind CD46 and SLAM (32). (B) Functional fusion formation assay of chimeric MV-H glycoproteins. After assessing expression of human (MDA-MB-468, HT-1080 and CASMC cells) and murine (NIH-3T3) uPAR by FACS (upper panel), cells were cotransfected with pCGF, a mammalian expression vector encoding MV-F (fusogenic) glycoprotein and either pTNHAALS-h-ATF (MDA-MB-468, HT1080 and CASMC) or pTNHAALS-m-ATF (NIH-3T3). Cell fusion was observed in the human (MDA-MB-231, HT1080) and murine (NIH-3T3) tumorigenic cells (arrows), but not in non-cancer cells (CASMC) 48 hours after cotransfection. Scale bar = 500 µm. After confirming functional activity of HAALS-ATF (human and murine), the chimeric glycoproteins were cloned individually into the PacI/SpeI sites of the measles virus genome (Fig. 1A). The human and murine retargeted viruses were named MV-h-uPA and MV-m-uPA, respectively. The enhanced green fluorescent protein (eGFP) gene was inserted into the Mlu/AatI site before the N gene. Fxa= factor Xa cleavage site (IEGR). H6= six-histidine peptide. N= nucleocapsid, P= phosphoprotein. M= matrix; F= fusion. H= hemagglutinin; L= polymerase gene. (C). I, RT-PCR analysis of MV-H glycoproteins in uPAR retargeted MVs (Left panel). Lane 1, MV-GFP; lane 2, chimeric MV-m-uPA; lane 3, MV- h-uPA. II, Immunoblot analysis of viral H protein using anti-H antibody. Equal titers of each virus were loaded. Lane 1, unmodified control H protein with mobility at 75 kDa; lane 2, chimeric MV-m-uPA; lane 3, MV- h-uPA. (D). In vitro viral propagation analysis of MV-h-uPA and MV-m-uPA by the one step growth curve in Vero-αHis cells. Titers (TCID50) of retargeted viruses were comparable to the unablated control virus (MV-GFP). I, Cell associated virus; II, cell released virus.