Adenovirus E3-6.7K protein is required in conjunction with the E3-RID protein complex for the internalization and degradation of TRAIL receptor 2

Abstract

Adenoviruses (Ads) encode several proteins within the early region 3 (E3) transcription unit that help protect infected cells from elimination by the immune system. Among these immunomodulatory proteins, the receptor internalization and degradation (RID) protein complex, which is composed of the RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K) subunits, stimulates the internalization and degradation of certain members of the tumor necrosis factor (TNF) receptor superfamily, thus blocking apoptosis initiated by Fas and TNF-related apoptosis-inducing ligand (TRAIL). The experiments reported here show that TRAIL receptor 2 (TR2) is cleared from the cell surface in Ad-infected cells. Virus mutants containing deletions that span E3 were used to show that the RID and E3-6.7K proteins are both necessary for the internalization and degradation of TR2, whereas only the RID protein is required for TRAIL receptor 1 downregulation. In addition, replication-defective Ad vectors that express individual E3 proteins were used to establish that the RID and E3-6.7K proteins are sufficient to clear TR2. These data demonstrate that E3-6.7K is an important component of the antiapoptosis arsenal encoded by the E3 transcription unit of subgroup C Ads.

FIG. 1.

Downregulation of TR2 in Ad-infected cells requires the RID and E3-6.7K proteins. HeLa cells were mock infected or infected with wt (rec700) or mutant viruses at a multiplicity of infection of 150 PFU/cell. At 23 h p.i., cells were detached from their dishes, stained with anti-TR2 (A) or anti-EGFR (B) antibody, and analyzed by flow cytometry. The virus used for infection (in parentheses) and the protein(s) deleted or mutated in that virus are shown to the right of each FACS profile.

FIG. 2.

Mutant Ads generally express equivalent amounts of E3 proteins. KB cells were mock infected or infected with wt (rec700) or mutant viruses at 100 PFU/cell. Proteins metabolically labeled with [³⁵S]cysteine were immunoprecipitated from cell lysates with anti-E3-6.7K or anti-RIDα antiserum. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by fluorography of the dried gel (top two panels). Both E3-6.7K (45) and RIDα (38) migrated as two bands when analyzed by SDS-PAGE. For Western blot analyses, HeLa cells were infected as described above. Proteins from cell lysates prepared at 24 h p.i. were separated by SDS-PAGE and then subjected to Western blot analysis with anti-RIDβ or anti-E3-14.7K antiserum (bottom two panels). RIDβ migrated as multiple bands (37) and E3-14.7K migrated as a doublet or a triplet (42) when analyzed by SDS-PAGE. The virus used for infection (in parentheses) and the protein(s) deleted or mutated in that virus are shown above each lane. The sizes of molecular weight standards (in thousands) are shown to the left of each gel or blot. The migration positions of the proteins detected are shown to the right of each gel or blot.

FIG. 3.

Internalization from the cell surface of TR2, but not TR1 or EGFR, requires both RID and E3-6.7K protein expression. HeLa cells were mock infected or infected with wt (rec700) or mutant viruses at 100 PFU/cell. At 17 h p.i., cells were fixed and then immunostained for TR2, TR1, or EGFR. The phenotype with respect to RID and E3-6.7K protein expression along with the name of the virus used for infection are shown.

FIG. 4.

TR1 and TR2 are degraded in Ad-infected cells. HeLa cells were mock infected or infected with wt or mutant viruses at 50 PFU/cell. Lysates were prepared at 26 h p.i., and a portion of each lysate was subjected to Western blot analysis to detect Fas, TfnR, Ad late proteins, and ERp72 (bottom four panels). In addition, a portion of each lysate was immunoprecipitated with anti-TR1 or anti-TR2 antibody prior to being subjected to Western blot analysis with anti-TR1 or anti-TR2 rabbit antiserum (top two panels). See the legend to Fig. 2 for additional explanations of designations.

FIG. 5.

Coinfection with E3-6.7K and RID protein-deficient viruses rescues the ability of Ads to downregulate TR2. A549 cells were mock infected or infected with wt (rec700) or mutant viruses at 150 PFU/cell. Note that each virus was used at 100 PFU/cell for coinfection (total of 200 PFU/cell). At 23 h p.i., cells were detached from their dishes, stained with a MAb against TR2 (A), TR1 (B), Fas (C), or TfnR (D), and analyzed by flow cytometry. The virus used for infection (in parentheses) and the protein(s) deleted or mutated in that virus are shown to the right of each FACS plot.

FIG. 6.

Maximal protection against TRAIL-induced apoptosis requires the expression of the E3-6.7K and RID proteins. HT29.14S cells were mock infected or infected with the indicated virus at 150 PFU/cell. Starting at 24 h p.i., cells were treated with TRAIL at 0, 0.5, 5.0, or 50.0 ng/ml plus CHX at 25 μg/ml for 24 h. Cell viability was determined by assaying for LDH release into the culture medium. The percent specific lysis was calculated as described in Materials and Methods and then plotted against the TRAIL concentration. Filled black circle, dl754 (gp19K⁻/6.7K⁻).

FIG. 7.

Infection with RD Ad vectors show that the E3-6.7K and RID proteins are sufficient for the downregulation of TR2. (A) HeLa cells were mock infected or infected with wt Ad (rec700) or Ad vectors that express different E3 proteins (Table 1). At 23 h p.i., cells were detached from their dishes, stained with anti-TR2 antibody, and analyzed by flow cytometry. (B) 293 cells were mock infected or infected at 20 PFU/cell with the vectors shown above the lanes. Note that each vector was used at 20 PFU/cell for coinfection. Proteins from cell lysates prepared at 24 h p.i. were separated by SDS-PAGE and then subjected to Western blot analysis with anti-E3-6.7K antiserum. The sizes of molecular weight standards (in thousands) are shown to the left of the blot. The migration positions of the two bands corresponding to E3-6.7K are shown to the right of the blot. (C) HeLa cells were mock infected or infected with 200 (Ad/RID and Ad/E3) or 300 (Ad/6.7K) PFU/cell. Note that Ad/RID at 200 PFU/cell and Ad/6.7K at 300 PFU/cell were used for coinfection. Following infection, cells were maintained in medium containing 1-β-d-arabinofuranosylcytosine (araC) in order to block cell division and to ensure that the infection did not progress from the early to the late stage. At 12-h intervals, the medium was replaced with medium containing fresh araC. At 48 h p.i., cells were detached from their dishes, stained with MAb against TR2, TR1, EGFR, or TfnR, and analyzed by flow cytometry.