Identification of Regions in the Moloney Murine Leukemia Virus SU Protein That Tolerate the Insertion of an Integrin-Binding Peptide

Abstract

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.

Change of Tropism of SL3-2 Murine Leukemia Virus, Using Random Mutational Libraries

“…3) are indicated. VRA, VRB, and VRC are defined according to sequence comparisons of the SL3-2 and ecotropic and amphotropic MLVs (37). PRR, the proline-rich region.…”

“…Attempts to change the tropism of a virus by engineering its RBD, either by insertion of ligands to known cell surface proteins (16,19,20,27,37) or by selection of randomized libraries (11), have had various degrees of success. Common to all of the previously engineered viral envelopes is that none of them was as efficient as the wild-type envelope.…”

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues

“…The first evidence of this limitation was observed with vectors carrying the Ram-1 phosphate transporter or the epidermal growth factor (EFG) into the GP, which showed a reduced transduction efficiency although the chimeric GPs were correctly expressed, processed, and exposed in the viral envelope (Cosset et al, 1995). A similar observation was reported for a gammaretroviral vector with a GP bearing a peptide binding to the α v β 3 integrin (Wu et al, 2000a), or with the stromal cell derived factor 1-alpha (Katane et al, 2002). These low transductions efficiencies were associated with loss or impairment of viral and cell surface proteins binding and subsequent membrane fusion and penetration of the viral core into the cytosol (Frecha et al, 2008;Lavillette et al, 2001;Yu & Schaffer, 2005).…”

Current and emerging treatments and surgical interventions for Morquio A syndrome: A review

“…The insertion of peptides did not affect the physical properties of the vector or infectious titers. [77][78][79] The resultant modified vector produced a viral capsid containing multiple copies of the D8 peptide (Figure 8). After intravenous infusion in a mouse model, the modified vector was targeted to bone, released from hydroxyapatite, transduced in bone cells, and expressed its gene product in bone markedly higher than in mice treated with unmodified vector (unpublished data).…”