Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein - PubMed (original) (raw)
Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein
S Snitkovsky et al. Proc Natl Acad Sci U S A. 1998.
Abstract
TVA, the cellular receptor for subgroup A avian leukosis viruses (ALV-A) can mediate viral entry when expressed as a transmembrane protein or as a glycosylphosphatidylinositol-linked protein on the surfaces of transfected mammalian cells. To determine whether mammalian cells can be rendered susceptible to ALV-A infection by attaching a soluble form of TVA to their plasma membranes, the TVA-epidermal growth factor (EGF) fusion protein was generated. TVA-EGF is comprised of the extracellular domain of TVA linked to the mature form of human EGF. Flow cytometric analysis confirmed that TVA-EGF is a bifunctional reagent capable of binding simultaneously to cell surface EGF receptors and to an ALV-A surface envelope-Ig fusion protein. TVA-EGF prebound to transfected mouse fibroblasts expressing either wild-type or kinase-deficient human EGF receptors, rendered these cells highly susceptible to infection by ALV-A vectors. Viral infection was blocked specifically in the presence of a recombinant human EGF protein, demonstrating that the binding of TVA-EGF to EGF receptors was essential for infectivity. These studies have demonstrated that a soluble TVA-ligand fusion protein can mediate viral infection when attached to specific cell surfaces, suggesting an approach for targeting retroviral infection to specific cell types.
Figures
Figure 1
The TVA-EGF protein. (A) A model of TVA-EGF mediated ALV-A infection. TVA-EGF is comprised of the extracellular domain of TVA fused via a proline-rich linker region to the mature form of human EGF (see Materials and Methods). TVA-EGF was predicted to bind to ALV-A SU ENV and to cell surface EGFRs, promoting viral entry. (B) TVA-EGF was produced in the extracellular supernatants of transfected human 293 cells. Samples of extracellular supernatants taken from 293 cells that were either not transfected (−) or transfected with plasmid DNA encoding TVA-EGF (+) were subjected to SDS/PAGE. After transfer to a nitrocellulose membrane, the TVA-EGF protein was detected by immunoblotting with an ALV-A SU-Ig fusion protein (11) and a horseradish peroxidase-coupled secondary antibody followed by enhanced chemiluminescence (Amersham). Molecular mass markers are given in kDa.
Figure 2
TVA-EGF binds specifically to the ligand-binding regions of cell surface EGFRs. (A) TVA-EGF binds in a saturable manner to M5 and to T23 cells. B82 mouse L cells lacking EGFRs, M5 cells expressing kinase-deficient human EGFRs, or T23 cells expressing wild-type human EGFRs (12, 13), were incubated with the indicated amounts of extracellular supernatants that contained TVA-EGF. The cells were then incubated with SUA-rIgG (11) and with fluorescein isothiocyanate-conjugated antibodies specific for rabbit Igs and analyzed by flow cytometry. Mean fluorescence values obtained with each cell population are shown. (B) EGF competes for TVA-EGF binding to M5 cells. M5 cells were incubated with extracellular supernatants containing TVA-EGF and increasing amounts of a recombinant human EGF protein. The level of cell surface-associated TVA-EGF in each cell population was measured by flow cytometry as described above. The data shown is representative of two independent experiments that were each performed in triplicate. (C) TVA-EGF disappeared more rapidly from the surfaces of T23 as opposed to M5 cells. M5 and T23 cells were preincubated at 4°C with extracellular supernatants that either lacked or contained TVA-EGF and then transferred to 37°C for the indicated periods of time. The TVA-EGF proteins that remained on the cell surface at each time point were detected by flow cytometry as described above. The data shown is representative of results obtained from three independent experiments.
Figure 3
The TVA-EGF protein mediates ALV-A infection when attached to cell surface EGFRs. (A) B82 and M5 cells incubated with medium that contained or lacked TVA-EGF, and T23TVA cells expressing a transmembrane form of TVA, were challenged with the subgroup A specific RCASH-A virus encoding hygromycin B phosphotransferase (7). The numbers of hygromycin B-resistant colonies, representing individual infection events, were counted and the results of independent experiments, each performed in triplicate, are shown. (B) B82, T23 and M5 cells were incubated with medium containing the indicated amounts of TVA-EGF and then challenged with the subgroup A-specific RCASA-Neo virus encoding neomycin phosphotransferase (2). The number of infected G418-resistant colonies was determined. T23TVA cells were also challenged with this virus, and the average number of infected G418-resistant colonies was determined after subtracting the small number of drug-resistant colonies that arose without virus addition (see Materials and Methods). These data are representative of three independent infection experiments that were each performed in triplicate. (C) EGF blocks TVA-EGF-dependent viral entry. M5 cells were incubated with extracellular supernatants containing TVA-EGF in the presence of different amounts of recombinant EGF and then challenged with the RCASA-Neo virus. The resulting G418-resistant infected colonies were counted, and the average numbers from two independent experiments that were each performed in triplicate are shown.
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