Human adenovirus 2 (Ad2) encodes an E3 11,600 MW protein named the """"""""Adenovirus Death Protein"""""""" (ADP). ADP is a nuclear (NM) glycoprotein synthesized in large amounts at late stages of infection. ADP colocalizes with Ad2 E1B-19K, a homolog of Bcl-2 which maintains cell viability. ADP may supersede the ability of E1B-19K to inhibit cell death; therefore, cells lyse and release virus. Structure-function analyses have identified domains required for ADP to promote cell death and be sorted from the ER to the Golgi and the NM where ADP functions to promote cell death. We propose to determine if ADP alone promotes cell death, it abrogates the ability of E1B-19K to inhibit cytolysis by TNF or anti-Fas, it affects the functions of the ICE family (which induces death), and it kills cells by apoptosis. The 'death' domains of ADP will be mutated to gain insight into how they function. Protein-protein interactions will be examined by coimmunoprecipitation using antisera to ADP and epitope tagged ADP, affinity binding to a GST- ADP fusion protein, and the yeast two-hybrid system. Cellular proteins that bind ADP will be cloned and characterized. We will focus on whether ADP binds to E1B-19K, cellular proteins that bind to E1B-19K, and to members of the Bcl-2 family. We will determine whether ADP localizes to the inner or outer NM, then use existing mutants to identify in transient transfections the domains in ADP necessary for transport of ADP from the trans-Golgi network to the NM, and for the interaction of ADP with E1B-19K. A CD8-ADP reporter will be used to identify sequences in ADP sufficient for targeting to the NM. ADP will be monitored by confocal immunofluorescence and by equilibrium density centrifugation of membrane fractions. ADP is a novel protein, and it represents a new concept in viral pathogenesis. ADP should be a powerful tool to understand apoptosis and the function of the E1B-19K oncoprotein, and perhaps members of the Bcl-2 family. ADP might also be used as a 'suicide' gene to kill cancer cells.

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