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 down-regulates 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 YXX Phi 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 122YXX Phi motif in 14.5 and the dileucine motif Leu 87-Leu88 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 Tyr122 in 14.5 is essential for rapid endocytosis of the 10.4-14.5 complex, whereas the 10.4LL motif acts down-stream 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.
D033961:Adaptor Protein Complex 1 / Q000378:metabolism; D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D017503:Adenovirus E3 Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D017209:Apoptosis; D045744:Cell Line, Tumor; D017124:Conserved Sequence; D015536:Down-Regulation; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D021382:Protein Sorting Signals; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism*; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D014162:Transfection; D019014:fas Receptor / Q000378:metabolism
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D017503:Adenovirus E3 Proteins; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; D019014:fas Receptor; D066246:ErbB Receptors