Identification of adenovirus type 12 candidate transformation proteins by radioimmunoprecipitation with antisera to EcoRI-C-Fragment transformed cells

Abstract

Experiments were performed to identify polypeptides coded by early gene block 1 (which includes the transforming region) of human adenoviruses in group A (Ad12, 18, 31). Two lines, C-1 and C-2, of rat cells transformed by transfection with Ad12 EcoRI-C fragment (left 16% of genome) were inoculated into syngeneic rats to produce tumors (F. Graham and S. Mak, unpublished data). The tumor sera were used to immunoprecipitate [ 35S]methi-onine-labeled polypeptides from Adl2-early-infected human cells. The polypeptides were resolved by electrophoresis in sodium dodecyl sulfate -polyacrylamide gels, and visualized by fluorography. Two additional sera were also used, from hamsters bearing tumors induced by inoculation with Ad12 or Ad18 virions. The following polypeptides were immunoprecipitated by the four sera: C-1-serum: 46,000 daltons (46K), 44K, 43K, 40K; C-2-serum: 46K, 44K, 43K, 40K, 16.5K, 10.5K; Adl2- and Ad18-sera: 65K, 60K, 55K, 54K, 47K, 45K, 11K, 10.5K. The sequence relationships between these polypeptides were investigated by the partial proteolysis procedure using Staphylococcus aureus V8 protease. The data suggested the following conditions: (1) The C-1- and C-2-specific 46K, 44K, 43K, and 40K are highly related. (2) The Ad12- and Ad18-specific 65K, 60K, 55K, 54K, 47K, and 45K are highly related. (3) The C-1- and C-2-specific 40–65K may be related to, but not identical with, the Ad12- and Ad18-specific 45–65K. (4) All three 10.5Ks are highly related. (5) Both 11Ks are highly related, and probably are unrelated to 16.5K or 10.5K. The immunoprecipitation results suggest (but do not prove) that early gene block 1 of group A Ads may code a “family” of related polypeptides with apparent molecular weights ranging from 40K-46K or 40K–65K, as well as polypeptides of 16.5K and 10.5K. One or more of these polypeptides may play a role in the initiation and/or maintenance of cell transformation.