Targeting of retroviral vectors to specific cells has been attempted through engineering of the surface (SU) protein of the murine leukemia viruses (MuLVs), but in many cases this has adversely affected protein function and targeted delivery has been difficult to achieve. In this study, we have inserted a 15-mer peptide that binds specifically to the alpha(v)beta(3) integrin into the Moloney MuLV SU protein, including regions that are surface exposed in the crystal structure of the ecotropic receptor-binding domain. We have concentrated in particular on the variable regions VRA, VRB, and VRC, which are responsible for the use of distinct cellular receptors by different MuLV subtypes and therefore may be more likely to accommodate a heterologous binding moiety. Despite these considerations, only 8 of 26 insertion sites were tolerated, including two separate regions in VRA, a cluster of sites in VRC, and previously identified sites at the N-terminus of the protein and in the proline-rich region immediately downstream of the receptor-binding domain. When expressed on retroviral vector particles, all of the viable proteins retained the ability to bind to and transduce murine cells, although the VRC mutants and an insertion in VRA gave reduced binding and titer. Finally, although all of the viable chimeras could bind to alpha(v)beta(3) in a solid-phase binding assay, we were unable to demonstrate expanded tropism for alpha(v)beta(3)-expressing human cells. This study highlights the difficulty of engineering the Moloney MuLV SU protein, even when structural information is available, and provides guidelines for the insertion of peptide ligands into the SU protein.
Copyright 2000 Academic Press.