FUNCTIONAL COMPLEMENTATION OF THE ADENOVIRUS E1B 19-KILODALTON PROTEIN WITH IN THE INHIBITION OF APOPTOSIS IN INFECTED-CELLS

Expression of the adenovirus E1A oncogene induces apoptosis which impe des both the transformation of primary rodent cells and productive ade novirus infection of human cells. Coexpression of E1A with the E1B 19, 000-molecular-weight protein (19K protein) or the Bcl-2 protein, both of which have antiapoptotic activity, is necessary for efficient trans formation. Induction of apoptosis by E1A in rodent cells is mediated b y the p53 tumor suppressor gene, and both the E1B 19K protein and the Bcl-2 protein can overcome this p53-dependent apoptosis. The functiona l similarity between Bcl-2 and the E1B 19K protein suggested that they may act by similar mechanisms and that Bcl-2 may complement the requi rement for E1B 19K expression during productive infection. Infection o f human HeLa cells with E1B 19K loss-of-function mutant adenovirus pro duces apoptosis characterized by enhanced cytopathic effects (cyt phen otype) and degradation of host cell chromosomal DNA and viral DNA (deg phenotype). Failure to inhibit apoptosis results in premature host ce ll death, which impairs virus yield. HeLa cells express extremely low levels of p53 because of expression of human papillomavirus E6 protein . Levels of p53 were substantially increased by E1A expression during adenovirus infection. Therefore, E1A may induce apoptosis by overridin g the E6-induced degradation of p53 and promoting p53 accumulation. St able Bcl-2 overexpression in HeLa cells infected with the E1B 19K(-) m utant adenovirus blocked the induction of the cyt and deg phenotypes. Expression of Bcl-2 in HeLa cells also conferred resistance to apoptos is mediated by tumor necrosis factor alpha and Fas antigen, which is a lso an established function of the E1B 19K protein. A comparison of th e amino acid sequences of Bcl-2 family members and that of the E1B 19K protein indicated that there was limited amino acid sequence homology between the central conserved domains of E1B 19K and Bcl-2. This doma in of the E1B 19K protein is important in transformation and regulatio n of apoptosis, as determined by mutational analysis. The limited sequ ence homology and functional equivalency provided further evidence tha t the Bcl-2 and E1B 19K proteins may possess related mechanisms of act ion and that the E1B 19K protein may be the adenovirus equivalent of t he cellular Bcl-2 protein.