Adenovirus type 2 early polypeptides immunoprecipitated by antisera to five lines of adenovirus-transformed rat cells

Abstract

We have identified adenovirus type 2 (Ad2)-induced early polypeptides (EPs) and have attempted to determine which EPs are coded by each of the four early gene blocks. [35S]methionine-labeled EPs were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cycloheximide pretreatment followed by labeling in hypertonic medium (210 to 250 mM NaCl) facilitated the detection of EPs. Seven major (reproducible bands in autoradiograms) EPs were detected with molecular weights of 74,000 (74K), 21K, 19K, 15K, 13.5K, 11.5K, and 11K. Minor (weaker bands) EPs of 55K, 52K, 42K, 18K, 12K, 8.8K, and 8.3K were also often seen. To identify and map the genes for virus-coded EPs, we prepared antisera against five lines of adenovirus-transformed cells that retain different fractions of the viral genome. The lines were F17, 8617, F4, and T2C4 transformed by Ad2 virions and 5RK (clone I) transformed by transfection with the Ad5 HsuI-G fragment (map position 0 to 8). The early gene blocks retained and expressed (in part) as RNA in these cells were as follows: 5RK(I), block 1 (70% of left 8% of genome); F17, block 1; 8617, blocks 1 and 4; F4 blocks 1, 2, and 4; T2C4, blocks 1, 2, 3, and 4. The following major EPs were immunoprecipitated: 15K by all antisera; 53K and 14.5K by F17, T2C4, 8617, and F4 antisera; 11.5K by T2C4, 8617, and F4 antisera; 44K, 42K, 19K, and 13.5K by T2C4 antisera; 11K by 8617 antisera. Minor EPs of 28K, 18K, and 12K were precipitated by all antisera except 5RK(I). The 53K and 15K EPs were precipitated also from Ad2 early infected monkey cells by the F17 antiserum and by sera from hamsters bearing tumors induced by Ad1-simian virus 40. The relationships between some of the immunoprecipitated EPs were investigated by the partial proteolysis procedure. All 53K EPs are the "same" (i.e., highly related), all 15K EPs are the "same," and all 11.5K EPs are the "same." The 15K EP is highly related to the 14.5 K EP. Although less certain, all 28K EPs appeared related, as did all 18K EPs. The T2C4-specific 44K EP is probably a dimer of the 21K glycopolypeptide. The T2C4-specific 13.5K EP and the 8617-specific 11K EP appear unrelated to any other polypeptides. These immunoprecipitation data provide evidence that early gene block I (map position 1 to 11) may encode major 53K, 15K, and 14.5K polypeptides, and minor 28K, 18K, and 12K polypeptides, and that all or some of the gene for 15K and 14.5K lies within map position 1 to 8. The surprisingly complex pattern of polypeptides coded by early gene block I raises the possibility that some polypeptides may be coded by overlapping "spliced" mRNA's. The possible block locations of the genes for the 21K, 13.5K, and 11.5K polypeptides are discussed.