Previous studies have examined targeting of pseudotyped MLV to human cells by using several strategies including antibody-streptavidin complexes that bridge the virus to target cell receptors (7, 21), display of variable antibody fragments as antibody-envelope fusion proteins (4, 22, 24, 31), and insertion of target ligands into wild-type envelope (5, 11, 13, 16, 25, 26-28). Typically, these modifications were at the N terminus of envelope, and viruses bearing such modified envelope derivatives were unable to transduce cells. These relatively large N-terminal modifications are believed to prevent envelope from undergoing the conformational change required for fusion and cell entry (31). The lack of full activity of N-terminally modified envelope derivatives necessitates the coexpression of wild-type envelope for production of transducing virus.

Integrin receptors are heterodimers composed of α and β subunits that play essential roles in cell-cell and cell-extracellular matrix interactions. Integrin receptor ligands contain the tripeptide RGD. RGD peptides as short as six amino acids (GRGDSP) can bind to integrin receptors and inhibit binding of full-length adhesion proteins (3, 6, 9, 29). Here we show that short RGD peptide ligands inserted at multiple locations within MLV envelope can direct an MLV retrovirus vector to transduce human cells. In addition, we demonstrate that the length and particular position of the inserted ligand can affect viral tropism.

We constructed >40 chimeric envelope derivatives containing in-frame insertions of either a 13- or a 21-amino-acid RGD peptide (RGD₁₃ or RGD₂₁, respectively; Table ​Table1).1). The core of the RGD₁₃ ligand is a six-amino-acid peptide, GRGDSP, which represents an RGD consensus sequence. The core of the RGD₂₁ ligand is a 14-amino-acid sequence, QGATFALRGDNPQG, derived from the mouse laminin protein (3). Both the RGD₁₃ and RGD₂₁ peptides were flanked by cysteine residues to constrain the sequence within a loop (3, 12, 29), as cyclization of RGD peptides has been shown to increase affinity for integrin receptors (20). In some cases, chimeric envelope derivatives with multiple ligands in tandem were also generated. Several of the chimeric envelope derivatives had deletions of envelope sequences, in addition to ligand insertions, as a result of multiple restriction enzyme cleavages. In all, 26 chimeric envelope derivatives containing the RGD₁₃ ligand, 16 chimeric envelope derivatives containing the RGD₂₁ ligand, and 5 chimeric envelope derivatives containing an RGE₂₁ ligand, a control nonbinding peptide (3, 10, 12, 23), were constructed.

Pseudotyped virus containing chimeric envelope derivatives (RGD viruses) was generated by using a human 293T-cell-based packaging cell line, Anjou 65 (18). Immunoblotting of purified virions indicated that in all cases tested the chimeric envelope derivatives were incorporated into the virion and correctly processed (data not shown). The RGD viruses were initially tested for their ability to transduce mouse NIH 3T3 cells and, on the basis of these results (Fig. ​(Fig.11 and ​and2),2), we can draw several conclusions. First, many of the RGD viruses retained their ability to transduce mouse cells, but those bearing insertions within the N terminus (RGD₁₃-4,5 and RGD₂₁-2,3), the VRA (RGD₁₃-8,12 and RGD₂₁-5), and the C-terminal region (RGD₁₃-19,23,34 and RGD₂₁-15,16) did not. Several of these latter RGD viruses also failed to transduce human cells (RGD₁₃-12,19,23,24 and RGD₂₁-5,15,16), whereas for others (RGD₁₃-4,5,8 and RGD₂₁-2,3) the defect was mouse cell specific. Second, most RGD₂₁ viruses transduced NIH 3T3 cells with efficiencies comparable to those of the equivalent RGD₁₃ viruses, and none of the RGD₂₁ viruses transduced NIH 3T3 cells with greater efficiency than the equivalent RGD₁₃ virus.

The RGD viruses were next tested for transduction of A375 human melanoma cells, which have been previously used to study integrin receptor binding (2, 8, 19). As expected, viruses bearing unmodified MLV envelope failed to transduce this human cell line. Significantly, however, many of the RGD viruses were able to transduce A375 human melanoma cells (Fig. ​(Fig.11 and ​and2).2). Transduction occurred when the RGD peptide was inserted at the N terminus (RGD₁₃-1-3 and RGD₂₁-1), within the N-terminal region (RGD₁₃-4-6 and RGD₂₁-2,3), within the VRA region (RGD₁₃-7,8,10,11 and RGD₂₁-4,6,7), and upstream of the PRR (RGD₁₃-14,15 and RGD₂₁-8,9). RGD viruses with insertions in the PRR and C-terminal region failed to transduce human cells. Several of the RGD viruses that transduced human cells failed to transduce NIH 3T3 cells (RGD₁₃-4,5,8 and RGD₂₁-2,3), indicating that viral tropism had been switched. In all cases tested, RGD viruses that transduced A375 human melanoma cells also transduced other human and nonhuman cell lines that contained integrin receptors (data not shown).

To examine the basis and specificity of human cell transduction, two experimental approaches were undertaken. First, the RGD₂₁ ligand was replaced with the equivalent RGE₂₁ sequence. Pseudotyped virus expressing an RGE₂₁ chimeric envelope derivative transduced NIH 3T3 host cells with efficiencies comparable to the equivalent RGD₂₁ derivative; in contrast, transduction of A375 human melanoma cells was significantly reduced (Fig. ​(Fig.3).3). Second, we analyzed the effect of antibodies to integrin receptors on the transduction of RGD viruses. NIH 3T3 and A375 human melanoma cells were pretreated with integrin receptor antibodies, and transduction was performed with two of the RGD₂₁ viruses. Transduction of human but not mouse cells was substantially reduced (Fig. ​(Fig.44).

We have developed a strategy for altering the host range of ecotropic retrovirus vectors by using chimeric envelope derivatives bearing short peptide ligands. In many instances, pseudotyped virus expressing a chimeric envelope derivative transduced human cells without removal of the N-terminal region or coexpression of wild-type envelope. Ligand insertions at multiple locations within the N-terminal half of envelope enabled transduction of human cells. In contrast, transduction of human cells did not occur with RGD peptide insertions in the PRR or C-terminal region of envelope, although many of these viruses could transduce mouse cells. Some of the RGD viruses bearing insertions at the N terminus or VRA region (RGD₁₃-4,5,8 and RGD₂₁-2,3) transduced human but not mouse cells. Collectively, these observations indicate that the position of the inserted ligand can dictate tropism.