Transduction of human cells by GRP virus. NIH 3T3 cells, human A375 melanoma cells, and human MDA-MB-231 breast carcinoma cells were infected with a GRP virus, selected with G418 for 2 weeks, fixed, and stained with Giemsa, and the colonies were counted. Amphotropic (Amph) and ecotropic virus (Eco) was generated by expressing the wild-type amphotropic and ecotropic envelopes, pCAA and pCEE, respectively. Note the log scale. The amphotropic envelope, pCAA, and the LAPNL packaging vectors were generated as described elsewhere (12); the latter expresses the secreted alkaline phosphatase (SEAP) gene and the neomycin resistance gene.

Transduction of NIH 3T3 cells and MDA-MB-453 breast carcinoma cells by HRG viruses. (A) NIH 3T3 cells, MDA-MB-453, and MDA-MB-231 breast carcinoma cells were infected with an HRG virus, and transduction was analyzed as described in the legend to Fig. 1. (B) Antibodies to HER3 and HER4 receptors block transduction of human cells by HRG viruses. NIH 3T3 and MDA-MB-453 breast carcinoma cells were pretreated with antibodies to HER3 and HER4 receptors (Lab Vision Corporation) and infected with the HRG-1 or HRG-8 virus, and transduction was analyzed as in Fig. 1.

Requirement of the GRP receptor for transduction of human cells by GRP viruses. (A) Antibodies to GRP block transduction of human cells by GRP viruses. GRP-1 or GRP-2 viruses were pretreated with 2A11 antibody (provided by Frank Cuttitta). NIH 3T3, A375 human melanoma cells, or MDA-MB-231 breast carcinoma cells were then infected with 2A11 antibody-treated GRP or untreated virus, and transduction was analyzed as in Fig. 1. The 2A11 antibody was added to pseudotyped virus at a 1:100 dilution, followed by incubation at 4°C for 4 h and then viral infection. (B) Requirement of the GRP receptor for transduction of human 293 cells. 293-GRPR-Zeo cells were infected with the GRP-1 or GRP-4 virus, with or without preincubation with the 2A11 antibody, and transduction was analyzed as in Fig. 1. The GRPR-Zeo construct was generated by insertion of the GRP receptor gene (GRP-R) (provided by James F. Battey, National Institutes of Health) into pcDNA3.1/Zeo+ (Invitrogen). 293-GRPR-Zeo cells were generated by transfection of 293 kidney cells with GRPR-Zeo, selection with zeocin, and verification of GRP receptor expression by reverse transcription-PCR. (C) Requirement of the GRP receptor for transduction of mouse cells by GRP-2, GRP-3, and GRP-5 viruses. NIH 3T3 and Swiss 3T3 cells were infected with a GRP virus, and transduction was analyzed as in Fig. 1.

Inducible destruction of human cancer cells by infection with a GRP virus and ganciclovir treatment. A375 human melanoma cells and MDA-MD-231 human breast carcinoma cells were infected with GRP-1 virus expressing either the SEAP or TK gene. Cells were selected with G418 for 2 weeks, followed by isolation of colonies and culture in media containing ganciclovir. The cell densities in the photographs are representative of the entire plate. The packaging vector, LTKNL, was generated by removal of the SEAP gene from LAPNL and insertion of the TK gene (provided by Steve Jones, University of Massachusetts Medical School). GRP virus with the LTKNL packaging construct was generated and used to transduce human cells. Colonies were isolated, grown in 10 μg of ganciclovir (Moravek Biochemicals, Inc.)/ml, and examined by using a Zeiss Axiophot microscope.