Evidence for involvement of BH3-Only proapoptotic members in adenovirus-induced apoptosis

Mammalian cells infected with human adenoviruses (Ads) undergo an apoptotic response as a result of expression of the viral E1A proteins, and this process is suppressed by the viral E1B-19K protein. The intermediary steps in the Ad-induced apoptosis pathway are not fully resolved. The apical step in the canonical mammalian apoptosis pathway involves functional activation of one or more of the BH3-only BCL-2 family proapoptotic proteins. Previous reports have suggested that Ad-induced apoptosis may be initiated at checkpoints downstream of the BH3-only proteins. Here, we undertook genetic and biochemical studies to determine the roles of BH3-only proteins in Ad-induced apoptosis. We examined the activities of the cellular antiapoptosis protein BCL-xL and its mutants expressed from the E1B region of the Ad5 genome. Our results showed efficient suppression of Ad-induced apoptosis by a BCL-xL mutant (mt1) deficient in interaction with multidomain proapoptotic proteins BAX and BAK but proficient in interaction with BH3-only proteins, suggesting a role for BH3-only proteins in the initiation of Ad-induced apoptosis. Further, the antiapoptotic activity of BCL-xL mt1 in Ad-infected cells was observed in spite of BAK activation as a consequence of MCL-1 degradation. Analysis of the mRNA levels of various BH3-only members by reverse transcription-PCR revealed prominent activation of the Bik gene. Further, the BIK protein was also modified into an apoptotically enhanced phosphorylated form during the viral infection. In addition to BIK, enhanced level of BIM was observed in Ad-infected cells. Between the two major EIA proteins coded by the 12S and 13S mRNAs, the 13S product appeared to contribute to the activation of these BH3-only members and apoptosis during viral infection. Depletion of BIK by the use of small interfering RNA reduced the level of Ad-induced apoptosis. Our results are consistent with a model that activation of the BH3-only members may initiate Ad-induced apoptosis.