Abstract
The 19-kDa glycoprotein (gp 19K) coded by early region E3 of adenovirus is of interest as a model for glycoprotein processing and sorting, as well as for the interaction between viral antigens and class I transplantation antigens. In this paper, we show that gp 19K is a major protein synthesized during early stages of infection of human KB cells. We report the purification of gp 19K to near homogeneity, the preparation of a gp 19K antiserum, and structural analyses on the protein. We have determined the DNA sequence of the gp 19K gene in adenovirus type 5 (Ad5) for comparison with the published sequence (Hérissé, J., Courtois, G., and Galibert, F. (1980) Nucleic Acids Res. 8, 2173-2192) of adenovirus type 2 (Ad2). Fragments produced by cyanogen bromide cleavage of Ad2 gp 19K are in accord with the DNA sequence, as are synthetic peptide antibodies targeted to the NH2 terminus of Ad2 gp 19K and the COOH terminus of Ad5 gp 19K. The Ad2 and Ad5 proteins are quite homologous. Conserved features include an NH2-terminal signal sequence, two potential Asn-linked glycosylation sites, and a 20-residue putative transmembrane hydrophobic domain followed by a 15-residue polar domain at the COOH terminus. We show that cleavage of the signal peptide occurs between the 17th and 18th amino acids on both the Ad2 and Ad5 versions of gp 19K and that both potential sites are glycosylated with exclusively high-mannose (as opposed to complex) oligosaccharides. Secondary structure predictions suggest six alpha-helix regions including the signal peptide and transmembrane domain, two or three beta-sheet regions, and about eight beta-turns including the two glycosylation sites and the regions flanking the transmembrane domain.
Highlights
From the tlnstitute for Molecular Virology and 7The Edward A
The 19-kDa glycoprotein coded by early tein, whichwe designate E3A-gpl9K or gp19Kis,’o~f ~interest region E3 of adenovirus is of interest as a model for as a model to understand glycoprotein processing [4,5,6] as glycoprotein processingand sorting, aws ell as for the well as the interactionbetweenviralantigensand class I
Its M, is lower when accord withthe DNA sequence, as are synthetic peptide translated in vitro in the presence of dog pancreas microsomes antibodies targeted to thNeHz terminus of Ad2 gpl9K than in their absence,suggesting that it undergoespostand the COOH terminus of Ad5 gpl9K
Summary
Ad2-induced Early ProP SJMet or [2-3HJMantwo or three&sheet regions, and about eight &turns. Two labeling procedures were employed Procedure i, in the presence of 1-j3-D-arabinofuranosylcytosinwehich maintains the cells in the early phase of infection by preventing the synthesis of viral DNA (lanesa and b); and Procedure ii, as in Procedure i but with cycloheximide pretreatment which enhances the levels of viral early mRNAs [34, 35] and the yield of viral early proteins(lanesc and d ) [21]. Both procedures have been used in experiments described in this paper.
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