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. 2004 Dec;78(23):13113-21.
doi: 10.1128/JVI.78.23.13113-13121.2004.

Inhibition of tumor necrosis factor (TNF) signal transduction by the adenovirus group C RID complex involves downregulation of surface levels of TNF receptor 1

Shawn P Fessler  1 , Y Rebecca Chin, Marshall S Horwitz
Affiliations

Inhibition of tumor necrosis factor (TNF) signal transduction by the adenovirus group C RID complex involves downregulation of surface levels of TNF receptor 1

Shawn P Fessler et al. J Virol. 2004 Dec.

Abstract

Adenoviruses employ multiple genes to inhibit the host antiviral responses. There is increasing evidence that these immunoregulatory genes may function either during lytic or latent infection. Adenovirus early transcription region 3 (E3) encodes at least seven proteins, five of which block the acquired or innate immune response. Previous findings from this laboratory demonstrated that the E3 proteins 10.4K and 14.5K, which form a complex in the plasma membrane, inhibit tumor necrosis factor (TNF)-induced activation of NF-kappaB and the synthesis of chemokines. To determine the mechanism of inhibition of these pathways by the adenovirus E3 10.4K/14.5K proteins, we have examined the effects of this viral complex on the inhibition of AP-1 and NF-kappaB activation by TNF and found a reduction in assembly of the TNF receptor 1 (TNFR1) signaling complex at the plasma membrane accompanied by downregulation of surface levels of TNFR1.

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Figures

FIG. 1.
FIG. 1.
TNF activation of NF-κB and AP-1 through TNFR1. Upon ligand binding, TNFR1 trimerizes, leading to signalosome formation and activation of both NF-κB and AP-1 transcription factors in parallel. Assembly of TRADD, TRAF-2, and RIP at the receptor is required for recruitment and activation of the IKK complex, which in turn phosphorylates IκB-α, signaling its polyubiquitination and proteasomal degradation. The p65/p50 heterodimer NF-κB is then able to translocate into the nucleus and bind its nuclear targets (2,6). For AP-1 activation, the assembly of TRADD and TRAF-2 at the receptor upon ligand-induced trimerization results in the recruitment and activation of the mitogen-activated protein kinases MEKK1, MKK4 or MKK7, and JNK, which phosphorylates and activates nuclear c-Jun of the Jun/Fos heterodimer comprising AP-1. This simplified diagram omits a number of interacting molecules that can affect the activities of the pathways shown.
FIG. 2.
FIG. 2.
RID is necessary and sufficient for the inhibition of TNF activation of NF-κB and AP-1. (A) 293 cells were infected with 4,000 particles/cell of Ad/RID or Ad/null. At 16 h. postinfection, cells were treated with 20 ng/ml TNF for 15 or 30 min or left untreated prior to harvest. Whole-cell extracts were resolved by SDS-PAGE and analyzed by Western blot with antibodies against IκB-α and β-tubulin. (B) HeLa cells were infected with 1,000 particles/cell of Ad/RID, Ad-CMV-GFP, or mock infected (M). At 16 h. postinfection, cells were treated with 20 ng/ml TNF for 20 min or left untreated prior to harvest. Whole-cell extracts were analyzed as in panel A. (C) Extracts from panel B were analyzed for the presence of activated c-Jun with an antibody against the phosphorylated residue at serine 63. Membranes were then stripped and reprobed for total amounts of c-Jun. (D) HeLa cells were infected with 1,000 particles/cell of Ad/RID, the E3 mutant dl309, which is deficient in RID and 14.7K, or 500 particles/cell of each virus. Cells were treated with TNF and analyzed for activated c-Jun as in panel C, or for degradation of IκB-α as in panels A and B. The increased amounts of c-Jun in cells infected with dl309 alone or in combination with Ad/RID indicate an E1-dependent increase in c-Jun, which did not affect the interpretation of the results. β-Tubulin was used as a protein loading control.
FIG. 3.
FIG. 3.
RID is sufficient for the inhibition of TNF activation of the NF-κB-dependent reporter construct pIgκ-Luc. (A) 293 cells were infected for 22 h with 1,000 particles/cell of Ad/RID or Ad/null. Cells were stimulated with TNF (20 ng/ml) or left untreated for 6 h. Luciferase activity was measured and normalized on the basis of Renilla luciferase (RL) activity. (B) 293 cells were transfected with either 400 ng of pMT2-RID-α plus 400 ng of pMT2-RID-β or 800 ng of empty plasmid with 100 ng of pIgκ-Luc and 10 ng of pRL-Tk; 18 h posttransfection, cells were treated with TNF or left untreated for 6 h. Luciferase activity was measured and normalized on the basis of Renilla luciferase activity.
FIG. 4.
FIG. 4.
RID inhibits TNFR1 signalosome formation. HeLa cells were infected with 1,000 particles/cell of Ad/RID or Ad/null for 18 h, then treated with 20 ng/ml TNF for 10 min or left untreated. Cells were lysed and extracts were immunoprecipitated with an anti-TNFR1 antibody. Resulting proteins were resolved by SDS-PAGE and analyzed by Western blot with antibodies against RIP, IKK-α, and IKK-β.
FIG. 5.
FIG. 5.
Flow cytometry analysis of surface TNFR1 in 293 and HeLa cells. 293 (A) or HeLa (B) cells were infected in triplicate with 4,000 particles/cell of Ad/RID, Ad/null, or mock infected, and stained for surface TNFR at 16 h (A) or 18 h (B) postinfection. Plots of surface TNFR1 fluorescence of representative samples are shown.
FIG. 6.
FIG. 6.
Kinetics of downregulation of receptors and inhibition of TNF-induced degradation of IκB-α. 293 cells were infected with 4,000 particles/cell of Ad/RID or Ad/null virus. At 8 or 12 h postinfection, surface levels of TNFR1 (A, D) or Fas (B, E) were measured by flow cytometry as in Fig. 5. Cells infected in parallel with Ad/RID or Ad/null were treated for 30 min with TNF (10 ng/ml) or left untreated. Cells were harvested at 8 h (C) or 12 h (F) postinfection and analyzed for IκB-α levels by Western blot.
FIG. 7.
FIG. 7.
Multiplicity of infection requirements for RID's downregulation of receptors. 293 cells were infected with various amounts of Ad/RID (3, 10, 30, or 100 PFU/cell) or 30 PFU/cell of Ad/null. Cells were analyzed for surface levels of TNFR1 or Fas, and the resulting geometric mean fluorescence intensities were plotted as percentages of geometric mean fluorescence obtained from Ad/null-infected cells (Ad/null fluorescence = 100, isotype control fluorescence = 0).
FIG. 8.
FIG. 8.
Total amounts of TNFR1 and Fas are reduced in the presence of RID. 293 cells were infected with increasing amounts of Ad/RID or Ad/null (100, 500, or 1,000 particles/cell) or mock infected. Cells were harvested at 20 h postinfection. Whole-cell extracts were resolved by SDS-PAGE and analyzed by Western blot with antibodies against TNFR1, Fas, and RID-β. β-Tubulin was analyzed as a protein loading control.
FIG. 9.
FIG. 9.
Hypertonic sucrose inhibition of RID-induced receptor downregulation. For either Fas or TNFR1 staining, 293 cells were infected in duplicate with 4,000 particles/cell of Ad/RID or Ad/null. At 4 h postinfection, medium for one well of Ad/RID or Ad/Null-infected cells was changed to Dulbecco's modified Eagle's medium with 10% fetal bovine serum containing 0.5 M sucrose. Sixteen hours postinfection, cells were harvested and surface levels of Fas (A) and TNFR1 (B) were determined by FACS. Fas staining of sucrose-treated Ad/null-infected cells was identical to that of sucrose-treated Ad/RID-infected cells (data not shown).
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