Abstract

Human adenoviruses (Ads) cause a variety of acute diseases but also establish persistent infections. Ads have evolved a number of mechanisms to evade the host immune response that seem to facilitate virus replication and transmission in vivo. Interestingly, one Ad transcription unit, the early transcription unit 3 (E3), appears to encode exclusively proteins with immuno-modulatory functions. Recently, a novel open reading frame, E3/49K, was identified in the E3 region of the epidemic keratoconjunctivitis causing serotype Ad19a. The sequence predicts a type I transmembrane protein with a large N-terminal domain and a short cytoplasmic tail of 19 amino acids (aa). The N-terminal part contains three repeat regions (R1-3) of about 80 aa each with R3 exhibiting a significant homology to immunoglobulin-like domains. This study shows that 49K is a highly glycosylated protein with an apparent molecular mass of 80-100 kDa. 12-13 out of 14 predicted N-glycosylation sites are utilized, and there is also evidence for O-glycosylation. 49K contains intramolecular disulfide bonds and is proteolytically processed. As a result of the cleavage the N-terminal ectodomain is secreted: Ad19a E3/49K is the first Ad protein known to be secreted. At steady-state, E3/49K is detected in the Golgi/TGN, early endosomes and at the cell surface of Ad19a-infected as well as 49K-transfected cells. The cytoplasmic tail, which contains two potential endosomal/lysosomal targeting motifs, YXXF and LL, plays a major role in the 49K trafficking. The proteolytic processing may occur in the TGN or secretory vesicles and/or at the cell surface. The finding that an increased cell surface expression of 49K correlates with an increased secretion rate indicates proteolytic processing of 49K at the cell surface. The C-terminal fragments and also unprocessed 49K proteins are internalized possibly by the interaction of the LL motif in the cytoplasmic tail with the clathrin adaptor complex AP-2. From early endosomes 49K proteins and C-terminal fragments recycle to the TGN potentially involving the interaction of the cytoplasmic tail of 49K with the clathrin adaptor complex AP-1 or to the cell surface, whereas a fraction is delivered to late endosomes and lysosomes. Remarkably, the secreted form of 49K specifically binds to various lymphocyte cell lines, including NK, B and T cell lines, but not to other cell types, e.g. fibroblasts, suggesting that the soluble and possibly also the membrane-bound 49K binds to receptor(s) on lymphocytes. Indeed, in Jurkat (T cell leukemia) cells, 49K-binding protein(s) with apparent molecular masses of 172-196 kDa were identified. Moreover, purified 49K inhibited the NK cell-mediated lysis of target cells. Thus, upon binding to cell surface receptors, 49K may trigger an inhibitory signal or inhibit an activating signal. While other Ad E3 proteins affect the infected cell, 49K seems to influence infiltrating cells of the immune system. Therefore, 49K function appears to represent an entirely novel Ad immune evasion mechanism.