Targeting avian leukosis virus subgroup A vectors by using a TVA-VEGF bridge protein - PubMed (original) (raw)

Targeting avian leukosis virus subgroup A vectors by using a TVA-VEGF bridge protein

S Snitkovsky et al. J Virol. 2001 Feb.

Abstract

Previously, we have demonstrated that bridge proteins comprised of avian leukosis virus (ALV) receptors fused to epidermal growth factor (EGF) can be used to selectively target retroviral vectors with ALV envelope proteins to cells expressing EGF receptors. To determine whether another type of ligand incorporated into an ALV receptor-containing bridge protein can also function to target retroviral infection, the TVA-VEGF110 bridge protein was generated. TVA-VEGF110 consists of the extracellular domain of the TVA receptor for ALV subgroup A (ALV-A), fused via a proline-rich linker peptide to a 110-amino-acid form of vascular endothelial growth factor (VEGF). This bridge protein bound specifically to its cell surface receptor, VEGFR-2, and efficiently mediated the entry of an ALV-A vector into cells. These studies indicate that ALV receptor-ligand bridge proteins may be generally useful tools for retroviral targeting approaches.

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Figures

FIG. 1

Construction and expression of retroviral receptor-ligand bridge proteins. (A) Recombinant genes encoding each bridge protein were generated by PCR-based methods and introduced into the pCI-plasmid expression vector (Promega) as shown. The numbering schemes for the amino acid residues of TVA, TVBS3, and heregulinβ1 were taken from references and and GenBank accession number B43273, respectively. The VEGF110 residues are described under GenBank accession number A44881. The positions of a proline-rich hinge region (PPPELLGGP) and of a 2-amino-acid insertion (His-Gly) that resulted during the construction of the TVBS3-containing bridge proteins are indicated. The TVA-VEGF110 monomer was expected to have a molecular mass ranging from 33 to 52 kDa because the primary amino acid sequence predicts a 22.4-kDa protein but the extracellular domain of TVA is subjected to extensive posttranslational modifications which add an additional 21 to 30 kDa to its apparent molecular mass (1, 2). Monomeric forms of TVBS3-VEGF110 and TVBS3-herβ1 were expected to have molecular masses of 37 and 33 kDa, respectively, based on their primary amino acid sequences (28 and 24 kDa, respectively) and the presence of three putative N-linked glycosylation sites in each protein. (B and C) Production of bridge proteins. Forty-five-microliter aliquots of extracellular supernatant taken from transfected human 293 cells that expressed the bridge proteins, or from nontransfected cells (negative controls), were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under either nonreducing (B) or reducing (C) conditions. The proteins were then transferred to a nitrocellulose membrane and were probed with subgroup A-specific (panel B) or subgroup B-specific (panel C) SU-rIg fusion proteins and then with a horseradish peroxidase-conjugated secondary antibody, as described previously (3, 23). The bridge proteins were then detected by enhanced chemiluminescence.

FIG. 2

TVA-VEGF110 binds specifically to cells that express VEGFR-2. PAE-VEGFR-2 cells (A) and PAE cells (B) were incubated with increasing amounts of a TVA-VEGF110-containing extracellular supernatant. (C) Prior to incubation with TVA-VEGF110, PAE-VEGFR-2 cells were incubated with extracellular supernatant that contained either TVBS3-VEGF110, TVBS3-herβ1, or no TVB-ligand bridge protein. Following these treatments the TVA-VEGF110 protein that was bound to the cells was detected by flow cytometric analysis using a subgroup A-specific SU-rIg fusion protein and an FITC-conjugated secondary antibody as described previously (23). These experiments were performed three times with similar results, and results of a representative example are shown.

FIG. 3

TVA-VEGF110 mediates ALV-A infection when bound to VEGFR-2. (A) PAE-VEGFR-2 cells were incubated with increasing amounts of extracellular supernatant containing TVA-VEGF110 and then challenged with 5 μl of a 100-fold concentrated stock of RCASBP(A)-EGFP. The total number of infected cells obtained was then calculated by flow cytometry as described previously (24). The average data obtained from an experiment that was performed in triplicate are shown with standard deviations indicated with error bars. (B) PAE-VEGFR-2 cells were incubated with equivalent amounts of TVBS3-VEGF110 or TVBS3-herβ1 or with no TVB-ligand bridge protein, prior to the addition of 10 μl of TVA-VEGF110. The cells were then challenged with RCASBP(A)-EGFP as in panel A and analyzed by flow cytometry. The results of an experiment performed in triplicate are shown with standard deviations of the data indicated with error bars.

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Targeting avian leukosis virus subgroup A vectors by using a TVA-VEGF bridge protein - PubMed (original) (raw)