Phosphoproteins of adenovirus 2

Abstract

Analysis of [ 32P]orthophosphate-labeled extracts of adenovirus 2 (AD2)-infected HeLa cells revealed three major adenovirus-specific phosphorylated species, of molecular weights (MW) 100,000, 72,000, and 33,000. The 72,000 MW species became detectable as a phosphorylated entity at about 15 hr after infection, the 33,000 and 100,000 MW species, at about 20–30 hr after infection. A variety of chemical and enzymatic tests indicated that each of the 32P-labeled species was a phosphoprotein, with the label esterified to serine and threonine residues. In the 100,000 and 33,000 MW proteins, the 32P was associated primarily with serine residues; the 72,000 MW protein contained approximately equimolar amounts of phosphoserine and phosphothreonine. Digestion of each protein with trypsin, followed by two-dimensional fingerprint analysis, suggested that the 72,000 MW protein contains two, or possibly three, sites for phosphorylation and that the 100,000 MW protein may be multiply phosphorylated. Two of the phosphoproteins exhibited an apparent increase in molecular weight during a pulse/chase labeling experiment (32,000 to 34,000 MW; 70,000 to 74,000 MW) which may reflect the post-translational phosphorylation of those proteins. None of the three major phosphoproteins is a structural component of purified adenovirions. Subcellular fractionation of infected cell lysates with the detergents NP-40 and Tween 40-sodium deoxycholate revealed that the 100,000 MW protein is located primarily in the cytoplasm, the 72,000 MW protein primarily in the nucleus, and the 33,000 MW protein in the nuclear membrane. The subcellular distribution of each protein was identical whether the proteins were labeled with [ 32P]orthophosphate or with radioactive amino acids. Analysis of the tryptic peptides of 14C-labeled amino acid-labeled proteins showed that many of the 14C-labeled tryptic peptides of the 33,000 MW protein were also present in the 100,000 MW protein. Why two such closely related proteins are sequestered in disparate regions of the infected cells is not known. Although 32P-labeled Ad2 structural proteins could not be visualized above the background of host phosphoproteins in a crude lysate, electrophoretic analysis of purified adenovirus virions revealed one major phosphorylated protein, of molecular weight 66,000 (Protein III a). With long exposures of the fluorograms, it was possible to detect nine additional phosphorylated virion products. Four of these (molecular weights 54,000, 39,000, 29,000 and 19,000) were present at low levels in the virion and have not previously been described. Subfractionation of the virions with pyridine revealed that two of these previously undescribed proteins (54,000 and 19,000 MW) are located in the DNA-containing core of the virion particle. The other five phosphoproteins [core V (48,500 MW), VI (24,000 MW), VII (18,500 MW), IX (12,000 MW), and XI (6000 MW)] were present in greater quantities, but only a small fraction of each of the molecules was phosphorylated.