Adenovirus RIDβ Subunit Contains a Tyrosine Residue That Is Critical for RID-Mediated Receptor Internalization and Inhibition of Fas- and TRAIL-Induced Apoptosis

Abstract

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RID␣ and RID␤ subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RID␤ that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RID␤ were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RID␤ C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxx). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RID␤ but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.

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

“…2B and 7). The importance of the equivalent residue (Tyr 124 ) in Ad5 14.5 was noted in a study using a heterologous overexpression system in COS7 cells aimed at identifying residues involved in signal transduction (25). The authors speculated that Tyr 124 may have a function in endocytosis although trafficking of 14.5 was not investigated.…”

“…The two 10.4 species form a disulfide-linked dimer via a conserved cysteine residue at position 31 (24) and associate non-covalently with the 14.5 protein in the ER (3). 14.5 is a type I transmembrane protein and the Ad5 product has been shown to be O-glycosylated and serine-phosphorylated (3,25). Both proteins seem to be exposed on the plasma membrane (26), but a thorough quantitative assessment is lacking.…”

Conditionally Replicating Adenoviruses Kill Tumor Cells via a Basic Apoptotic Machinery-Independent Mechanism That Resembles Necrosis-Like Programmed Cell Death

“…Ads have a rather complex infrastructure in which the viral genes produce numerous proteins that prevent the death of infected cells early after infection and other proteins that favor cell death at later stages (6,28,33,38,42,52). Several viral encoded proapoptotic and antiapoptotic proteins are known to maintain temporal control of the Ad on the host cell.…”

Development of transcriptionally regulated oncolytic adenoviruses

“…Long considered dispensable for viral replication in vitro, more recent studies have suggested an important role for the E3 region, in vivo (Ilan et al, 1997;Yu et al, 1999a). The E3 proteins function as modulators of the immune response and, in later stages of the viral life cycle, assist in host cell lysis and spread of progeny virions (Gooding et al, 1988(Gooding et al, , 1991Krajcsi and Wold, 1992;Hermiston et al, 1993;Tollefson et al, 1996;Lichtenstein et al, 2002Lichtenstein et al, , 2004. Therefore, deletion of some or all of these genes may result in an increased immune response that could enhance destruction of the tumor mass.…”