Figure 2. RHVP pQa-1 is GPI anchored, cell surface expressed and assembles with β2m.

(A) Schematic depiction of the pQa-1 expression constructs used in the study. The C-terminal 26aa containing predicted GPI attachment site (marked by red star) is shown under the C-terminus of the last construct. (B) Mouse embryonic fibroblast (MEF) and human 293 T cells were stably transduced with the vector only or pQa-1-HA construct depicted in (A). Surface expression of pQa-1 on these cells was analyzed by flow cytometry using anti-HA antibody. (C) Left panel: cells were treated with (blue) or without (red) 0.069 U/ml phosphatidylinositol-specific phospholipase C (PI-PLC) at 37°C for 45 min before staining with anti-HA or anti-L^d (30-5-7). MEFs expressing vector only served as background staining (solid gray). The representative of two independent experiments is shown. Right panel: following incubation with indicated concentration of PI-PLC, MEF cells expressing L^d-pQa-1 or Thy1.1 were examined. Here endogenous MHC-I (H2–K^b) serves as a negative control protein; its level of surface expression was unaffected by PI-PLC. (D) Following a 30-min pulse with ³⁵S-Cys/Met, pQa-1 transduced MEF cells were lysed with 1% NP-40 and immunoprecipitated for pQa-1 using anti-HA. The precipitated proteins were resolved on SDS-PAGE and visualized by autoradiography (left) or immunoblotted with the indicated antibodies (right). The representative of two independent experiments is shown. (E) MHC-Ia- and β2m-deficient MEFs (H2-K^b-/- H2-D^b-/- B2m^-/-; 3KO) or 3KO+β2m cells transduced with pQa-1-HA or vector control were examined for surface pQa-1 expression using anti-HA. The representative of two analyses is shown.