FIG 5.

UL148A's downregulation of MICA is functional, and UL148A targets MICA to lysosomal degradation. (A) FLS3 MICA*004-HA cells were mock infected or infected with BAC2 or the BAC2 ΔUL148A mutant. NK cells were incubated 48 hpi with a blocking anti-NKG2D MAb or with no antibody and then incubated with the aforementioned HFFs. Data represent results from two independent experiments. Error bars represent SEM. Student's t test was performed to evaluate significance. *, P < 0.05; **, P < 0.005; N.S., nonsignificant. (B to E) FLS3 MICA*004-HA cells were mock infected (M) or infected with BAC2 (B) or with BAC2 ΔUL148A (Δ) and were incubated for 12 h with a vehicle-only treatment (Vehicle), lysosome inhibitors (in panel B, leupeptin [Leu], NH₄Cl, or concanamycin A [ConA]), or proteasome inhibitors (in panel C, epoxomicin [Epx] or MG132). Cells were lysed 48 hpi, and Western blotting was performed using anti-MICA antibody for detection and anti-vinculin antibody as a loading control. (D and E) Quantification of two independent lysosome (D) or proteasome (E) inhibition experiments. MICA protein levels were quantified relative to the loading control (Vinculin). RQ, relative quantification. Error bars represent SEM. Student's t test was performed to evaluate significance. *, P < 0.05. The ConA panel is derived from a separate gel with a vehicle-only control.