TRAF3IP3 Is Cleaved by EV71 3C Protease and Exhibits Antiviral Activity

Figures

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  Figure 1. TRAF3IP3 interacts with EV71 3Cpro. (A) HEK293T cells were co-transfected with GFP-3C-C147S and HA-TRAF3IP3. Cells were harvested 48 h after transfection and co-immunoprecipitated using anti-HA antibodies, and protein interactions were detected using Western blotting. WCL, whole-cell lysate. (B) HEK293T cells were co-transfected with HA-TRAF3IP3 and Myc-3C-C147S. Anti-Myc antibodies were used for co-immunoprecipitation, and other operations were the same as in (A). (C) HA-TRAF3IP3 or Myc-3C-C147S were transfected into HeLa cells. 48 h later, the cells were fixed and protein subcellular localizations were observed using immunofluorescence. Scale bars = 10 μm.

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  Figure 2. TRAF3IP3 is the cleavage substrate for 3Cpro. (A) HA-TRAF3IP3 or GFP was co-transfected with 3Cpro or C147S into HEK293T cells. Cells were harvested 48 h later for Western blotting. (B) HEK293T was co-transfected with HA-TRAF3IP3 and a gradient of Myc-3C, with C147S as a non-cleavable control. The cleavage bands were detected using the antibody recognizing the C-terminus of TRAF3IP3. (C) EV71 at an MOI of 10 was used to infect into Jurkat cells for 0, 4, 8, 12, and 24 h, and the cleavage of endogenous TRAF3IP3 was detected using Western blotting. * and ** represent short and long exposures, respectively.

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  Figure 3. The 87Q-88G is the only site in TRAF3IP3 that is cleaved by 3Cpro. (A) Schematic representation of the potential 3Cpro cleavage sites in TRAF3IP3. (B) HEK293T cells were transfected with HA-TRAF3IP3 1–301 or 302–551 and Myc-3C. Forty-eight hours later, Western blotting was used to detect the proteins. (C) HA-TRAF3IP3 wild-type or G88A mutant was co-transfected with Myc-3C or mutant C147S into HEK293T cells. Cells were harvested 48 h after transfection for Western blotting analysis. (D) HEK293T cells were transfected with HA-TRAF3IP3 wild-type or G88A mutant. Cells were harvested 48 h after transfection and lysates were incubated with purified 3Cpro or mutant 3C-C147S for 90 min at 30°C. Loading buffer was added to stop the reaction, followed by Western blotting detection of proteins. (E) HA-TRAF3IP3 single-mutant S303A or double-mutant G88A S303A (M2) were co-transfected with Myc-3C or mutant C147S into HEK293T cells. Cells were harvested 48 h after transfection for Western blotting. (F) HA-TRAF3IP3-S303A or M2 was transfected into HEK293T cells. The rest of the experiment was the same as the in vitro cleavage in (D).

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  Figure 4. TRAF3IP3 inhibits EV71 replication. (A) Jurkat cells were first infected with lentiviruses expressing negative control shRNA (shNC) or an shRNA targeting TRAF3IP3 (shTRAF3IP3). Cells were selected with puromycin until the knockdown effect stabilized. Jurkat cells were harvested for Western blotting at 12 h after infection with 1 MOI EV71. (B) Jurkat cells were first infected with lentiviruses expressing Flag-TRAF3IP3 or vector. The rest of the experiment was the same as (A). (C) HA-TRAF3IP3 wild-type or G88A mutant was transfected into RD cells, followed by infection with EV71 at 1 MOI at 36 h after transfection. Twelve hours later, the cells were collected for Western blotting.

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  Figure 5. Identification of the NLS and NES of TRAF3IP3. The subcellular localization of the protein was detected using immunofluorescence analysis. Cells were fixed 48 h after transfection. Scale bars represent 10 μm. (A) HeLa cells were transfected with HA-TRAF3IP3, HA-TRAF3IP3 1–87, or HA-TRAF3IP3 88–551. (B) Schematic representation of the pC3-EGFP-X-GST vector. HeLa cells were transfected with EGFP-X-GST vector, EGFP-SV40 NLS-GST, EGFP-TRAF3IP3 1–87-GST, or EGFP-TRAF3IP3 30–37-GST. (C) HeLa cells were transfected with HA-TRAF3IP3 or HA-TRAF3IP3 Δ30–37. (D) Schematic representation of the pC3-EGFP vector. HeLa cells were transfected with EGFP vector or EGFP-TRAF3IP3 407–416. (E) HeLa cells were transfected with HA-TRAF3IP3 or HA-TRAF3IP3 Δ407–416. (F) Schematic representation of the NLS and NES of TRAF3IP3.

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  Figure 6. The NES of TRAF3IP3 contributes to the inhibition of EV71. (A) HA-TRAF3IP3 wild-type or deletion mutants were transfected into RD cells, followed by infection with EV71 at an MOI of 1 at 36 h post-transfection. Twelve hours later, the cells were collected for Western blotting. (B) HA-TRAF3IP3 wild-type or deletion mutants and Myc-3C-C147S were co-transfected into HeLa cells. Cells were fixed for immunofluorescence after 48 h. Scale bars represent 10 μm.