MODIFICATIONS IN THE BINDING DOMAIN OF AVIAN RETROVIRUS ENVELOPE PROTEIN TO REDIRECT THE HOST-RANGE OF RETROVIRAL VECTORS

On the basis of theoretical structural and comparative studies of vari ous avian leukosis virus SU (surface) envelope proteins, we have ident ified four small regions (I, II, III, and IV) in their receptor-bindin g domains that could potentially be involved in binding to receptors. From the envelope gene of an avian leukosis virus of Subgroup A, we ha ve constructed a set of SU mutants in which these regions were replace d by the coding sequence of FLA16, a 16-amino-acid RGD-containing pept ide known to be the target for several cellular integrin receptors. He lper-free retroviral particles carrying a neo-lacZ retroviral vector w ere produced with the mutant envelopes. SU mutants in which regions II I and TV were substituted yielded normal levels of envelope precursors but were not detectably processed or incorporated in viral particles. In contrast, substitutions in regions I and II did not affect the pro cessing and the viral incorporation of SU mutants. When FLA16 was inse rted in region II, it could be detected with antibodies against FLA16 synthetic peptide, but only when viral particles were deglycosylated. Viral particles with envelopes mutated in region I or Il were able to infect avian cells through the subgroup A receptor at level similar to those of the wild type. When viruses with envelopes containing FLA16 peptide in region II were applied to plastic dishes, they were found t o promote binding of mammalian cells resistant to infection by subgrou p A avian leukosis viruses but expressing the integrins recognized by FLA16. Deglycosylated helper-free viruses obtained by mild treatment w ith N-glycosidase F have been used to infect these mammalian cells, an d infections have been monitored by neomycin selection. No neomycin-re sistant clones could be obtained after infection by viruses with wild- type envelopes. Conversely, colonies were obtained after infection by viruses with envelopes bearing FLA16 in region II, and the genome of t he retroviral vector was found correctly integrated in cell DNA of the se colonies. By using a blocking peptide containing the minimal adhesi ve,RGD sequence contained in FLA16, we have shown that preincubation o f target cells could specifically inhibit infection by viruses with FL A16.