Two distinct transport motifs in the adenovirus E3/10.4-14.5 proteins act in concert to down-modulate apoptosis receptors and the epidermal growth factor receptor

The adenovirus (Ad) early transcription unit E3 encodes immunosubversive functions. The E3 transmembrane proteins 10.4 and 14.5 form a complex that downregulates the epidermal growth factor receptor and apoptosis receptors from the cell surface by diverting them to endosomes/lysosomes for degradation. The latter process protects infected cells from ligand-induced apoptosis. The mechanism by which 10.4 - 14.5 mediate re-routing remains elusive. We examined the role of putative YXXPhi and dileucine (LL) transport motifs within Ad2 10.4 - 14.5 for target protein modulation. By generating stable E3 transfectants expressing 10.4 - 14.5 proteins with alanine substitutions in these motifs, we show that 3 of the 5 motifs are essential for functional activity. Whereas tyrosine 74 in 14.5 appears to be important for efficient 10.4 - 14.5 interaction, the (122)YXXPhi motif in 14.5 and the dileucine motif Leu(87)-Leu(88) in 10.4 constitute genuine transport motifs: disruption of either motif abolished binding to the cellular adaptor proteins AP-1 and AP-2, as shown by surface plasmon resonance spectroscopy, and caused missorting, dramatically altering cell surface appearance and the intracellular location of viral proteins. Fluorescence-activated cell sorter analysis and immunofluorescence data provide evidence that Tyr(122) in 14.5 is essential for rapid endocytosis of the 10.4 - 14.5 complex, whereas the 10.4LL motif acts downstream and protects 10.4 - 14.5 from extensive degradation by rerouting it into a recycling pathway. Infection of primary cells with adenoviruses carrying the relevant point mutations confirmed the crucial role of these transport motifs for down-regulation of Fas, TRAIL-R1, TRAIL-R2, and epidermal growth factor receptor. Thus, two distinct transport motifs present in two proteins synergize for efficient target removal and immune evasion.