(A) FLS3 HFFs transduced with an empty vector, MICA*004 or MICA*008, were either UI or infected at an MOI of 2–4 with the indicated virus and lysed 72 hpi. Western blot was performed using α-MICA antibody. α-GAPDH served as loading control.
(B) FLS1 and VH3 HFFs were either UI or infected at an MOI of 2–4 with the indicated virus and lysed 72 hpi. Western blot was performed using α-MICA antibody. α-GAPDH served as loading control.
(C) FLS3 HFF expressing MICA*008 were either UI or infected at an MOI of 2–4 with the indicated virus and lysed 72 hpi. The lysates were either untreated or digested with endoglycosidase H or with PNGase F (marked N, H, and F, respectively). Western blot was performed using α-MICA antibody. α-Vinculin served as loading control. Arrows indicate MICA forms with and without carbohydrates (CH+/−).
(D) Quantification of the 34-kDa GPI-anchored form of MICA*008 shown in (C), normalized according to the loading control and then normalized relative to the quantity in the UI control.
(E) VH3 HFFs were UI or infected at an MOI of 2–4 with the indicated virus and lysed at 72 hpi. The lysates were untreated or digested with endoH or with PNGaseF (marked N, H, and F, respectively). Western blot was performed using α-MICA. α-Vinculin served as loading control. Arrows indicate MICA forms with and without carbohydrates (CH+/−).
(F) Quantification of the 34 kDa GPI-anchored form of MICA*008 shown in (E), normalized according to the loading control and then normalized relative to the quantity in the UI control.
(G) A model of HCMV effect on MICA*008: (1) following HCMV infection, MICA*008 mRNA is upregulated and the protein is translated into the ER lumen. (2) MICA*008’s immature, nonanchored form remains in the ER for a prolonged period, and unknown HCMV protein(s) bind and sequester it at this stage. (3) MICA*008, which has escaped other HCMV mechanisms, undergoes GPI anchoring via an unknown noncanonical pathway. US9 targets this stage and diverts MICA*008 to the cytosol, where it is subsequently degraded by the proteasome. Viral mechanisms are marked by red borders.
See also Figure S6.