Inhibition of TNF signal transduction by the adenovirus RID complex
Chin, Yuet Ming Rebecca
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For productive acute infection and successful persistence in the host, adenoviruses have evolved to express a number of immuno-modulatory proteins in the E3 region. Our laboratory has demonstrated that the E3-RIDalpha/beta complex inhibits TNF-induced activation of NFkappaB. Experiments were undertaken to examine the mechanisms behind this effect. We showed, using flow cytometry, that RID down-regulates TNF receptor 1 (TNFR1) expression at the plasma membrane, thereby inhibiting TNF-induced cytosolic signal transduction, including the signalosome assembly at the receptor and IkappaBalpha degradation. To study the role of the RIDbeta tyrosine sorting motif in the removal of surface TNFR1, tyrosine122 on RIDbeta was mutated to alanine or phenylalanine. Not only did both RIDbeta mutations abolish the down-regulation of surface TNFR1, but they paradoxically increased surface TNFR1 levels. RID also down-regulates other death receptors such as FAS; however, surface FAS expression was not increased by RIDbeta mutants, suggesting that regulation of TNFR1 and FAS by RID are mechanistically different. In mixing experiments, the wild-type (WT) RID-mediated TNFR1 down-regulation was partially inhibited in the presence of RIDbeta mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDbeta and TNFR1 was shown by co-immunoprecipitation. In contrast, the mutants did not affect the WT RID-induced down-regulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS intracellularly. By using siRNA against the mu2 subunit of adaptor protein 2, a dominant negative dynamin construct K44A, and lysosomotropic agents, we also demonstrated the important role of clathrin in the down-regulation of TNFR1, and showed that the degradation of TNFR1 occurs via an endosomal/lysosomal pathway. Surface biotinylation experiments revealed that although RID and TNFR1 were able to form a complex on the cell surface, the rate of TNFR1 endocytosis was not affected by RID. However, the degradation of internalized TNFR1 was enhanced significantly in the presence of RID. Our data suggest that RID down-regulates TNFR1 levels by altering the fate of internalized TNFR1 that becomes associated with RID at the plasma membrane, likely by promoting its sorting into endosomal/lysosomal degradation compartments.
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