Abstract
Vaccinia virus growth factor (VGF), a highly glycosylated 77-residue epidermal growth factor (EGF)-like polypeptide encoded in vaccinia poxvirus, is reported to play an important role in stimulating growth of uninfected cells to facilitate virus infection. We have chemically synthesized the unglycosylated forms of VGF and VGF19-69, a shortened VGF analog consisting of 51 residues and comprising the EGF-homologous region (position 19-69) of VGF. Both synthetic forms of VGFs were purified to homogeneity and vigorously characterized by various criteria, including the Cf-252 ion fission fragment mass spectrometry, amino acid sequencing, and enzymatic digestion to confirm the disulfide linkages. Synthetic VGFs exhibited high affinity binding to the EGF receptors in A431, NRK 49F, NRK clone 3, and NIH 3T3 cells, but, unlike the glycosylated form, showed contrasting mitogenic activities in various cells in vitro. Synthetic VGFs showed low levels of mitogenic and colonogenic activities in NRK clone 49F cells and NIH 3T3 cells, full agonist activities in human keratinocytes and Swiss 3T3 cells, and partial agonist activities in NRK clone 3 cells. Our results suggest that the unglycosylated form of VGF is an EGF antagonist to selected cells and that the production of unglycosylated form of VGF by the cytolytic vaccinia virus may serve as a mechanism whereby inhibition of growth and metabolism of selected host cells may be used to facilitate the propagation of the virus infection.
Highlights
Vaccinia virus growth factor (VGF), a highly glycosylated 77-residue epidermal growth factor (EGF)-like polypeptide encoded in vaccinia poxvirus, is reported to play an important role in stimulating growth of uninfected cells to facilitate virus infection
In this article we show that VGF and VGF19-es, both synthesized chemically and unambiguously by the solid-phase method, are EGF/TGFa antagonists in NRK clone 49F and NIH 3T3 cells, agonists in human keratinocytes and Swiss
Synthesis and Characterization of VGF and VGF19-69-The chemical syntheses of VGF and VGF,9-69 were carried out manually by the conventional stepwise solid-phase approach (Merrifield, 1963, 1986) using a strategy employing differential acid-labile protecting groups of the tert-butyloxycarbonyl and benzyl alcohol type protecting groups, as used in our previous syntheses of TGFcv (Tam et al, 1984, 198613;Tam, 1987) and Shope fibroma growth factor (Lin et al, 1988)
Summary
Synthesis-The syntheses of VGF and VGF,,-,, were manually carried out in silanized reaction vessels by a stepwise solid-phase procedure (Merrifield 1963; Tam et aL., 1986b) on 1 g each of Boc-. The peptide resin (0.5 g) containing either VGF or VGF,,-G, was treated four times (10 ml each) with 1 M thiophenol in dimethylformamide for 8 h to remove the N’“-Dnp protecting group of His and with 50% trifluoroacetic acid in CH&l, for 20 min to remove the Boc group on the NH?. After dilution with 200 ml of 2 M urea, 0.1 M Tris/HCl buffer, the peptide solution containing 100 mg of methionine was treated with 100 mg of reduced glutathione and 200 mg of oxidized glutathione at room temperature for 36 h. Cells were incubated again with (100 J/well) DMEM containing 0.2% for NRK cells or 0.5% calf serum for NIH 3T3 and. Growth Inhibition Assays-Growth inhibition assays including [“HI thymidine assay and soft agar assay were carried out as described in previous experimental sections except that 1) the concentrations of EGF or TGFN were constant whereas concentrations of VGFs varied (Figs. 4, 5, and 7) or 2) the concentration of VGF or VGF,,mfis was constant whereas the concentration of EGF or TGFa varied (Fig, 6)
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