Adenovirus RIDalpha regulates endosome maturation by mimicking GTP-Rab7

Endocytosis serves many important functions ranging from acquisition of extracellular nutrients to regulation of cell surface receptor expression and signal transduction, maintenance of cell polarity, and antigen presentation. Many intracellular pathogens have evolved mechanisms to hijack endocytic pathways in order to invade cells, proliferate, and ensure pathogen survival. Understanding the consequences of pathogenic infection on endosomal membrane dynamics has enabled greater understanding of the intracellular trafficking machinery. In cases where the cellular targets are known, isolated pathogenic genes have also been used to curb pathological processes associated with these targets and treat human disease models. This study focused on an integral membrane protein encoded by the early region 3 of human adenoviruses called RIDalpha, which was originally identified because of its ability to divert constitutively recycling EGF receptors (EGFRs) to lysosomes. The specific aims of this study were to identify protein interactions of RIDalpha, demonstrate the importance of these protein interactions in RIDalpha activity, and gain a better understanding of the mechanism employed by RIDalpha to mediate receptor down-regulation. We report that RIDalpha binds to RILP and ORP1L, which are known effectors for the Rab7 GTPase that governs transport from early to late endosomes and then to lysosomes. RIDalpha localizes to endocytic vesicles but shares no appreciable sequence homology with Rab7 and does not possess intrinsic GTPase or other catalytic activity. We show that RIDalpha enhances late endocytic trafficking and compensates for reduced or dominant-negative Rab7(T22N) expression. In vitro, the interaction between RIDalpha and RILP was facilitated by Cu2+ binding to His75 and His76 in the RIDalpha cytosolic tail, and site-directed mutagenesis of these two residues resulted in loss of RIDalpha-RILP interactions in cells. The mutant RIDalpha failed to enhance late endocytic trafficking or to promote the cell surface clearance of EGFR and Fas. We additionally demonstrate cellular interleukin-8 release through a mechanism dependent on EGFR signaling, suggesting a potential benefit of EGFR cell surface clearance to adenoviral pathogenesis. We conclude that RIDalpha mediated down-regulation of cell surface receptors occurs through the recruitment of GTP-Rab7 effectors to compartments that would ordinarily be perceived as early endosomes, thereby promoting their microtubule dependent displacement towards lysosomes.