Redirecting retroviral tropism by insertion of short, nondisruptive peptide ligands into envelope

Abstract

A potentially powerful approach for in vivo gene delivery is to target retrovirus to specific cells through interactions between cell surface receptors and appropriately modified viral envelope proteins. Previously, relatively large (>100 residues) protein ligands to cell surface receptors have been inserted at or near the N terminus of retroviral envelope proteins. Although viral tropism could be altered, the chimeric envelope proteins lacked full activity, and coexpression of wild-type envelope was required for production of transducing virus. Here we analyze more than 40 derivatives of ecotropic Moloney murine leukemia virus (MLV) envelope, containing insertions of short RGD-containing peptides, which are ligands for integrin receptors. In many cases pseudotyped viruses containing only the chimeric envelope protein could transduce human cells. The precise location, size, and flanking sequences of the ligand affected transduction specificity and efficiency. We conclude that retroviral tropism can be rationally reengineered by insertion of short peptide ligands and without the need to coexpress wild-type envelope.

FIG. 1.

Transduction of NIH 3T3 cells and A375 human melanoma cells by RGD₁₃ viruses. NIH 3T3 and A375 human melanoma cells were infected with an RGD virus and then selected with G418 for 2 weeks, fixed, and stained with Giemsa, and the colonies were counted. The amphotropic virus, Amph, was generated by expressing the amphotropic envelope, pCAA, and the ecotropic virus, Eco, was generated by expressing the wild-type ecotropic envelope, pCEE. Note the log scale. The pCAA expression vector was generated by removing the amphotropic envelope gene from the full-length infectious clone(17) and insertion into pCEE after removal of the ecotropic envelope. The packaging construct, LAPNL, was generated by removal of the VSV-G envelope from LGRNL (30) and insertion of the secreted alkaline phosphatase gene (SEAP; Tropix). Pseudotyped virus producer cell lines were generated by cotransfection of Anjou 65 cells with LAPNL and a plasmid expressing a chimeric envelope derivative using Dotap (Boehringer), followed by selection in zeocin (200 μg/ml) for 2 weeks. RGD₁₃ required cotranfection of a zeocin expression plasmid (Invitrogen).

FIG. 2.

Transduction of NIH 3T3 cells and A375 human melanoma cells by RGD₂₁ viruses (as described for Fig. 1).

FIG. 3.

Requirement of the RGD sequence for transduction of human cells. NIH 3T3 (A) and A375 human melanoma (B) cells were infected with an RGD₂₁ or RGE₂₁ virus, and transduction was analyzed as described in the legend to Fig. 1.

FIG. 4.

Antibodies to integrin receptors block transduction of human cells. NIH 3T3 (A) and A375 human melanoma (B) cells were pretreated with polyclonal antibodies to β₁, β₃, and α_ν integrin receptors (Santa Cruz Biotechnology), infected with RGD₂₁-1, RGD₂₁-4, or RGD₂₁-9 viruses, and transduction was analyzed as described in the legend to Fig. 1. The three antibodies were diluted 1:100 in Dulbecco modified Eagle medium and incubated with cells for 4 h. Cells were then incubated with pseudotyped virus for 6 h.