Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins

Abstract

The T-cell-mediated immune response plays a central role in the defense against intracellular pathogens. To avoid this immune response, viruses have evolved elaborate mechanisms that target and modulate many different aspects of the host's immune system. A target common to many of these viruses is the major histocompatibility complex (MHC) class I molecules. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes K3 and K5 zinc finger membrane proteins which remove MHC class I molecules from the cell surface. K3 and K5 exhibit 40% amino acid identity to each other and localize primarily near the plasma membrane. While K3 and K5 dramatically downregulated class I molecules, they displayed different specificities in downregulation of HLA allotypes. K5 significantly downregulated HLA-A and -B and downregulated HLA-C only weakly, but not HLA-E, whereas K3 downregulated all four HLA allotypes. This selective downregulation of HLA allotypes by K5 was partly due to differences in amino acid sequences in their transmembrane regions. Biochemical analyses demonstrated that while K3 and K5 did not affect expression and intracellular transport of class I molecules, their expression induced rapid endocytosis of the molecules. These results demonstrate that KSHV has evolved a novel immune evasion mechanism by harboring similar but distinct genes, K3 and K5, which target MHC class I molecules in different ways.

FIG. 1

Downregulation of surface expression of MHC class I molecules by K3 and K5. BJAB cells were electroporated with GFP reporter, GFP-K3, GFP-K4.2, or GFP-K5 vector. The cell surface levels of MHC class I molecules were assessed 48 h posttransfection by staining the cells with a W6/32 antibody for MHC class I or TU39 antibody for MHC class II (y axis) and gating the GFP-positive cell population (x axis) by flow cytometry. Three populations of cells were distinguished by the applied gates; in the upper left quadrant are the untransfected cells, in the upper right quadrant are the GFP-positive transfected cells which have not downregulated class I or class II, and in the lower right quadrant are the GFP-positive transfected cells that have downregulated class I or class II. The data were reproduced in three independent experiments.

FIG. 2

Downregulation of MHC class I molecules on cells stably expressing K3 or K5. (A) Downregulation of MHC class I molecules by K3 or K5. G418-resistant cells were stained with a PE-conjugated W6/32 antibody or a PE-conjugated pan-class I antibody and analyzed by flow cytometry. Two hundred thousand events were collected on a FACScan flow cytometer. As a control, a histogram of each cell line (shaded) is overlaid with a dotted-line histogram of an isotype antibody control. The mean value of the relative level of class I and II surface expression is presented inside each graph. (B) Expression of K3 and K5. BJAB cells were labeled with [³⁵S]methionine and [³⁵S]cysteine overnight. Radioactive cell lysates were used for immunoprecipitation with an anti-six-His antibody. Lane EF, BJAB/EF; lane K3, BJAB/K3; lane K5, BJAB/K5. The arrows indicate the K3 and K5 proteins.

FIG. 3

Localization of K3 and K5 near the plasma membrane. (A) Localization in COS-1 cells. COS-1 cells were transfected with pEF-K3-6xHis (K3), pEF-K4.2-6xHis (K4.2), or pEF-K5-6xHis (K5) DNA. The cells were permeabilized with ethanol and reacted with a Texas red-conjugated anti-six-His antibody (red) and FITC-conjugated Golgi-specific dye (green). Immunofluorescence was examined with a Leica confocal immunofluorescence microscope. To determine their specific locations, stained cells were cross-sectioned and viewed under the Leica confocal immunofluorescence microscope and are presented in the side boxes. The overlapping staining was visualized as yellow color. (B) Localization in BJAB cells. BJAB/EF, BJAB/K3, and BJAB/K5 cells were permeabilized with ethanol and reacted with a Texas red-conjugated anti-six-His antibody. Immunofluorescence was examined with a Leica confocal immunofluorescence microscope.

FIG. 4

Endo H sensitivity of class I molecules. BJAB/EF, BJAB/K3, and BJAB/K5 cells were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine for 15 min and chased for 0, 20, or 40 min. Radioactively labeled lysates were used for immunoprecipitation with an anti-class I antibody. Immunoprecipitates were treated with endo H prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The portion of the gel displaying the heavy chain of class I molecules is shown. ∗, the endo H-resistant heavy chain of class I molecules.

FIG. 5

Rapid endocytosis of class I molecules by K3 and K5. BJAB/EF, BJAB/K3, and BJAB/K5 cells were stained with a FITC-conjugated class I antibody (A) and a Texas red-conjugated transferrin receptor antibody (B) at 4°C for 30 min, washed twice with cold culture medium, and incubated at 37°C to allow for the internalization of antibody-bound class I molecules and transferrin receptor. At the indicated time points (in minutes), the localization of class I and transferrin receptor was determined with a Leica confocal immunofluorescence microscope.

FIG. 6

Selective downregulation of HLA allotypes by K3 and K5. 221 cells stably expressing defined HLA allotypes were electroporated with GFP, GFP-K3, or GFP-K5 vector. Cell surface levels of MHC class I molecules were assessed 48 h posttransfection by staining the cells with PE-conjugated pan-class I W6/32 antibody (y axis) and gating the GFP-positive cell population (x axis) by flow cytometry. The numbers inside the boxes indicate the average mean values of class I surface expression of the GFP-positive cell population. The data were reproduced in three independent experiments.

FIG. 7

Downregulation of CD8 chimera containing transmembrane and cytoplasmic regions of HLA-A2 by K3 and K5. 221 cells stably expressing CD8α or the CD8/A2-Tm+tail chimera were electroporated with GFP reporter, GFP-K3, or GFP-K5 vector. The cell surface level of CD8 molecules was assessed 48 h posttransfection by staining the cells with an anti-CD8 antibody (y axis) and gating the GFP-positive cell population (x axis) by flow cytometry. The numbers inside the boxes indicate the average mean values of CD8 surface expression of the GFP-positive cell population. The data were reproduced in three independent experiments.

FIG. 8

Regions of class I molecules required for downregulation by K3 and K5. 221 cells stably expressing class I chimeric molecules were electroporated with GFP reporter, GFP-K3, or GFP-K5 vector. The level of MHC class I surface expression was assessed 48 h posttransfection by staining the cells with a PE-conjugated W6/32 antibody for class I (y axis) and gating the GFP-positive cell population (x axis) by flow cytometry. The numbers inside the boxes indicate the average mean values of MHC class I surface expression of the GFP-positive cell population. The data were reproduced in three independent experiments.