Fig. 4. NTRV sequence at the cytoplasmic region of K3 is required for MHC class I down-regulation. A schematic of the location of various mutations of the K3 protein is shown at the top. Five mutations, K3 ΔNTRV (ΔNTRV), K3 N₁₅₆/A (N/A), K3 T₁₅₇/A (T/A), K3 R₁₅₈/A (R/A) and GFP K3 V₁₅₉/A (V/A), were introduced into K3. Each mutant is described in greater detail in the text. Along the right side, the ability of wild-type K3 and each mutant to down-regulate MHC class I, as shown below the schematic, was given a relative score. ‘+++, ++, + and –’ indicate strong, weak, very weak and no activity of the K3 mutant in MHC class I down-regulation, respectively. The differential MHC class I down-regulation activity of several K3 mutants is shown below the schematic. To examine activity of K3 and its mutants in MHC class I down-regulation, BJAB cells were electroporated with pTracer–GFP–K3 (WT) or one of the K3 mutant vectors. Cell surface level of MHC class I was assessed 48 h post-transfection by staining for MHC class I (y axis) and gating the GFP-positive cell population (x axis) by flow cytometry. Numbers in the upper right side quadrant represent the MCF of MHC class I staining in the GFP-positive population, calculated as described in the legend to Figure 2. The data were reproduced in at least two independent experiments.