This application addresses two """"""""sets"""""""" of novel proteins encoded by the human adenovirus (Ad) E3 transcription unit. One """"""""set"""""""" of proteins consists of two integral membrane proteins named 10.4K (10,4001 kDa) and 14.5K, which function as the 10.4K/14.5K heterodimer. The other """"""""set"""""""" is the nonmembrane protein named 14.7K. The 10.4K/14.5K complex is multifunctional: it (i) inhibits tumor necrosis factor (TNF)-induced apoptosis and synthesis of arachidonic acid (AA) from cytosolic phospholipase A, (cPLA,), (ii) inhibits TNF-induced translocation of cPLA, from the cytosol to membranes (where cPLA, functions), (iii) downregulates Fas (a cell-surface receptor) and inhibits Fas-induced apoptosis, (iv) downregulates the epidermal growth factor receptor (EGFR), and (v) inhibits inflammation in Ad-infected mouse lungs. The proposed experiments will use a large collection of mutants, cell lines, vectors, and antisera to 10.4K/14.5K to understand the mechanism for these functions. One hypothesis is that 10.4K/14.5K is a """"""""shuttle complex"""""""" that contains motifs which regulate protein sorting and protein-protein interactions, thereby directing Fas and EGFR to lysosomes where they are degraded. The 14.7K protein also inhibits TNF-induced apoptosis and AA release. Experiments with 14.7K mutants, vectors, and cell lines will address if 14.7K inhibits ICE family proteases that function in apoptosis, and if 14.7K can inhibit apoptosis induced by agents other than TNF. Much effort will be directed to cloning and characterizing cellular proteins that bind to 10.4K, 14.5K, and 14.7K. The 14.7K protein inhibits inflammation in the lungs of Ad-infected mice and in a transgenic mouse expressing 14.7K in its lungs. The anti-inflammatory mechanisms of 14.7K and l0.4K/14.5K will be studied in the 14.7K-transgenic mouse and proposed 10.4K/14.5 K-transgenic mousc infected by various viruses. The 10.4K/14.5K and 14.7K proteins are proposed to function in Ad biology to inhibit TNF-, Fas-, and CTL-induced apoptosis, and to prevent TNF-induced inflammation^H These proteins provide unique insight^H into viral pathogenesis, TNF- and Fas-induced apoptosis and signal transduction, cPLA2 function, EGFR function, and protein sorting.
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