A Tyrosine-Based Signal Plays a Critical Role in the Targeting and Function of Adenovirus RIDα Protein

Abstract

Early region 3 genes of human adenoviruses contribute to the virus life cycle by altering the trafficking of cellular proteins involved in adaptive immunity and inflammatory responses. The ability of early region 3 genes to target specific molecules suggests that they could be used to curtail pathological processes associated with these molecules and treat human disease. However, this approach requires genetic dissection of the multiple functions attributed to early region 3 genes. The purpose of this study was to determine the role of targeting on the ability of the early region 3-encoded protein RID␣ to downregulate the EGF receptor. A fusion protein between the RID␣ cytoplasmic tail and glutathione S-transferase was used to isolate clathrin-associated adaptor 1 and adaptor 2 protein complexes from mammalian cells. Deletion and site-directed mutagenesis studies showed that residues 71-AYLRH of RID␣ are necessary for in vitro binding to both adaptor complexes and that Tyr72 has an important role in these interactions. In addition, RID␣ containing a Y72A point mutation accumulates in the trans-Golgi network and fails to downregulate the EGF receptor when it is introduced into mammalian cells as a transgene. Altogether, our data suggest a model where RID␣ is trafficked directly from the trans-Golgi network to an endosomal compartment, where it intercepts EGF receptors undergoing constitutive recycling to the plasma membrane and redirects them to lysosomes.Adenoviruses (Ads) are nonenveloped DNA viruses that replicate and assemble in the host cell nucleus (13). Ads are responsible for approximately 5% of acute upper respiratory tract (3) and 15% of lower respiratory tract (1) infections in infants and children. Similar to other DNA viruses, Ads are also capable of establishing persistent infections, because they have evolved numerous strategies to evade host antiviral surveillance mechanisms. Some of these same mechanisms also facilitate survival of the virus during an acute infection. Thus, a thorough understanding of viral genes that control host immune and inflammatory responses is fundamental to our ability to prevent and treat virus-induced disease at multiple levels. These mechanisms have also influenced strategies for designing Ad-based vectors for gene therapy (14).The early region 3 (E3) transcription genes of human Ads encode several proteins that exploit the intracellular trafficking machinery to modify host immune or inflammatory responses (11, 29). Thus, E3 proteins have served as novel probes of membrane transport mechanisms in addition to providing insights to Ad pathophysiology. The most abundant E3 protein, E3/19K, suppresses the host adaptive immunity response by retaining class I MHC molecules in the endoplasmic reticulum. Other E3 proteins affect the functions of molecules involved in proliferation and apoptosis, intracellular cell signaling events linked to NF-B, and secretion of proinflammatory chemokines. Two E3 proteins, RID␣ (also called E3-10.4 and E3-13.7) and RID␤ (also called E3-14.5),...

Adenovirus early region 3 RIDα protein limits NFκB signaling through stress-activated EGF receptors

“…Using an extensive series of adenovirus deletion mutants, we mapped the viral gene responsible for this effect to an E3 transcript encoding a small membrane protein called RIDα-C2 [48,49]. We have also shown that RIDα-C2 was a resident membrane protein in a novel population of endosomes, where it transiently interacted with EGFRs and re-routed them for degradation [19,50,51,52]. In contrast to the ligand-induced pathway, however, adenovirus-induced EGFR trafficking did not require intrinsic tyrosine kinase activity or receptor ubiquitination [19,53].…”

Adenovirus Modulates Toll-Like Receptor 4 Signaling by Reprogramming ORP1L-VAP Protein Contacts for Cholesterol Transport from Endosomes to the Endoplasmic Reticulum

“…A bacterial expression plasmid encoding glutathione S-transferase (GST) fused to the 30-amino-acid C terminus of RID␣ (RID␣-CT) has been described previously (56). BL21 cells were transformed with GST plasmids and cultured at 37°C to an optical density at 600 nm (OD 600 ) of approximately 0.6, induced with 0.1 mM isopropyl-␤-D-thiogalactopyranoside for 16 h at room temperature, and collected by low-speed centrifugation.…”

“…These mutations did not have a significant impact on RID␣ binding in an established pulldown assay with a glutathione S-transferase (GST) fusion protein encoding ORP1L binding sequences in the RID␣ C terminus (RID␣-CT) ( Fig. 4C) (56). The roles of FFAT and ORD-lid domains were evaluated in shNPC1-RID␣ cells treated with a 3=-untranscribed-region (3=-UTR)-specific ORP1L siRNA and then reconstituted with siRNA-resistant GFP-ORP1L constructs encoding the wild-type protein or mutant variants with (i) a D478A substitution in the FFAT motif that eliminates VAP binding; (ii) a 4-amino-acid deletion (Δ560 -563) that attenuates the sterol sensing capacity of the ORD-lid downstream of Rab7; and (iii) a dual D478A/ Δ560 -563 substitution ( Fig.…”

Evaluation of anti-EGFR-iRGD recombinant protein with GOLD nanoparticles: synergistic effect on antitumor efficiency using optimized deep neural networks

“…These sets of proteins cling to targeted phosphotyrosine residues and further boost intracellular signalling through numerous pathways. 35,36 3.6. Ras/Raf/mitogen-activated protein kinase pathway…”